Ebola virion proteins expressed from venezuelan equine encephalitis (VEE) virus replicons

ABSTRACT

Using the Ebola GP, NP, VP24, VP30, VP35 and VP40 virion proteins, a method and composition for use in inducing an immune response which is protective against infection with Ebola virus is described.

[0001] Ebola viruses, members of the family Filoviridae, are associatedwith outbreaks of highly lethal hemorrhagic fever in humans and nonhumanprimates. The natural reservoir of the virus is unknown and therecurrently are no available vaccines or effective therapeutic treatmentsfor filovirus infections. The genome of Ebola virus consists of a singlestrand of negative sense RNA that is approximately 19 kb in length. ThisRNA contains seven sequentially arranged genes that produce 8 mRNAs uponinfection (FIG. 1). Ebola virions, like virions of other filoviruses,contain seven proteins: a surface glycoprotein (GP), a nucleoprotein(NP), four virion structural proteins (VP40, VP35, VP30, and VP24), andan RNA-dependent RNA polymerase (L) (Feldmann et al. (1992) Virus Res.24, 1-19; Sanchez et al., (1993) Virus Res. 29, 215-240; reviewed inPeters et al. (1996) In Fields Virology, Third ed. pp. 1161-1176.Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds. Lippincott-RavenPublishers, Philadelphia). The glycoprotein of Ebola virus is unusual inthat it is encoded in two open reading frames. Transcriptional editingis needed to express the transmembrane form that is incorporated intothe virion (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93,3602-3607; Volchkov et al, (1995) Virology 214, 421-430. The uneditedform produces a nonstructural secreted glycoprotein (sGP) that issynthesized in large amounts early during the course of infection.Little is known about the biological functions of these proteins and itis not known which antigens significantly contribute to protection andshould therefore be used to induce an immune response.

[0002] Recent studies using rodent models to evaluate subunit vaccinesfor Ebola virus infection using recombinant vaccinia virus encodingEbola virus GP (Gilligan et al., (1997) In Vaccines 97, pp. 87-92. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), or naked DNAconstructs expressing either GP or sGP (Xu et al. (1998) Nature Med. 4,37-42) have demonstrated the protective efficacy of Ebola virus GP inguinea pigs. (All documents cited herein supra and infra are herebyincorporated in their entirety by reference thereto.) Additionally,Ebola virus NP and GP genes expressed from naked DNA vaccines(Vanderzanden et al., (1998) Virology 246, 134-144) have elicitedprotective immunity in BALB/c mice. However, successful vaccination ofnonhuman primates with individual Ebola virus genes has not beendemonstrated. Therefore, there exists a need for a vaccine which isefficacious for protection from Ebola virus infection.

SUMMARY OF THE INVENTION

[0003] The present invention satisfies the need discussed above. Thepresent invention relates to a method and composition for use ininducing an immune response which is protective against infection withEbola virus.

[0004] Because the biological functions of the individual Ebola virusproteins are not known and the immune mechanisms necessary forpreventing and clearing Ebola virus infection are not well understood,it was not clear which antigens significantly contribute to protectionand should therefore be included in an eventual vaccine candidate toinduce a protective immune response. We evaluated the ability ofpackaged Venezuelan equine encephalitis (VEE) virus replicons expressingGP, NP, VP40, VP35, VP30 and VP24 virion proteins of Ebola virus toelicit protective immunity in two strains of mice which differ at themajor histocompatibility locus. There are no published reports of the VPproteins having been assayed as antigens for the production of an immuneresponse in a mammal.

[0005] The VEE virus replicon (Vrep) is a genetically reorganizedversion of the VEE virus genome in which the structural protein genesare replaced with a gene from an immunogen of interest, such as theEbola virus virion proteins. This replicon can be transcribed to producea self-replicating RNA that can be packaged into infectious particlesusing defective helper RNAs that encode the glycoprotein and capsidproteins of the VEE virus. Since the packaged replicons do not encodethe structural proteins, they are incapable of spreading to new cellsand therefore undergo a single abortive round of replication in whichlarge amounts of the inserted immunogen are made in the infected cells.The VEE virus replicon system is described in U.S. patent to Johnston etal., U.S. Pat. No. 5,792,462 issued on Aug. 11, 1998.

[0006] For our purposes, each of the Ebola virus genes were individuallyinserted into a VEE virus replicon vector. The VP24, VP30, VP35, andVP40 genes of Ebola Zaire 1976 (Mayinga isolate) were cloned by reversetranscription of RNA from Ebola-infected Vero E6 cells and viral cDNAswere amplified using the polymerase chain reaction. The Ebola Zaire 1976(Mayinga isolate) GP and NP genes were obtained from plasmids alreadycontaining these genes (Sanchez, A. et al., (1989) Virology 170, 81-91;Sanchez, A. et al., (1993) Virus Res. 29, 215-240) and were subclonedinto the VEE replicon vector.

[0007] After characterization of the Ebola gene products expressed fromthe VEE replicon constructs in cell culture, these constructs werepackaged into infectious VEE virus replicon particles (VRPs) andsubcutaneously injected into BALB/c and C57BL/6 mice. As controls inthese experiments, mice were also immunized with a VEE repliconexpressing Lassa nucleoprotein (NP) as an irrelevant control antigen, orinjected with PBS buffer alone. The results of this study demonstratethat VRPs expressing the Ebola GP, NP, VP24, VP30, VP35 or VP40 genesinduced protection in mice and may provide protection in humans.

[0008] Therefore, it is one object of the present invention to provide aDNA fragment encoding each of the Ebola Zaire 1976 GP, NP, VP24, VP30,VP35, and VP40 virion proteins (SEQUENCE ID NOS. 1-7).

[0009] It is another object of the present invention to provide the DNAfragments of Ebola virion proteins in a recombinant vector. When thevector is an expression vector, the Ebola virion proteins GP, NP, VP24,VP30, VP35, and VP40 are produced.

[0010] It is yet another object of the present invention to provide aVEE virus replicon vector comprising a VEE virus replicon and a DNAfragment encoding any of the Ebola Zaire 1976 (Mayinga isolate) GP, NP,VP24, VP30, VP35, or VP40 proteins. The construct can be used as anucleic acid vaccine or for the production of self replicating RNA.

[0011] It is another object of the present invention to provide a selfreplicating RNA comprising the VEE virus replicon and any of the EbolaZaire 1976 (Mayinga isolate) RNAs encoding the GP, NP, VP24, VP30, VP35,and VP40 proteins described above. The RNA can be used as a vaccine forprotection from Ebola infection. When the RNA is packaged, a VEE virusreplicon particle is produced.

[0012] It is another object of the present invention to provideinfectious VEE virus replicon particles produced from the VEE virusreplicon RNAs described above.

[0013] It is further an object of the invention to provide animmunological composition for the protection of subjects against Ebolavirus infection, comprising VEE virus replicon particles containing theEbola virus GP, NP, VP24, VP30, VP35, or VP40 proteins, or anycombination of different VEE virus replicons each containing one or moredifferent Ebola proteins selected from GP, NP, VP24, VP30, VP35 andVP40.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

[0015]FIG. 1 is a schematic description of the organization of the Ebolavirus genome.

[0016]FIGS. 2A, 2B and 2C are schematic representations of the VEEreplicon constructs containing Ebola genes.

[0017]FIG. 3 shows the generation of VEE viral-like particles containingEbola genes.

[0018]FIG. 4 is an immunoprecipitation of Ebola proteins produced fromreplicon constructs.

DETAILED DESCRIPTION

[0019] In the description that follows, a number of terms used inrecombinant DNA, virology and immunology are extensively utilized. Inorder to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Filoviruses. The filoviruses(e.g. Ebola Zaire 1976) cause acute hemorrhagic fever characterized byhigh mortality. Humans can contract filoviruses by infection in endemicregions, by contact with imported primates, and by performing scientificresearch with the virus. However, there currently are no availablevaccines or effective therapeutic treatments for filovirus infection.The virions of filoviruses contain seven proteins: a membrane-anchoredglycoprotein (GP), a nucleoprotein (NP), an RNA-dependent RNA polymerase(L), and four virion structural proteins (VP24, VP30, VP35, and VP40)Little is known about the biological functions of these proteins and itis not known which antigens significantly contribute to protection andshould therefore be used in an eventual vaccine candidate.

[0020] Replicon. A replicon is equivalent to a full-length virus fromwhich all of the viral structural proteins have been deleted. A multiplecloning site can be inserted downstream of the 26S promoter into thesite previously occupied by the structural protein genes. Virtually anyheterologous gene may be inserted into this cloning site. The RNA thatis transcribed from the replicon is capable of replicating andexpressing viral proteins iri a manner that is similar to that seen withthe full-length infectious virus clone. However, in lieu of the viralstructural proteins, the heterologous antigen is expressed from the 26Spromoter in the replicon. This system does not yield any progeny virusparticles because there are no viral structural proteins available topackage the RNA into particles.

[0021] Particles which appear structurally identical to virus particlescan be produced by supplying structural protein RNAs in trans forpackaging of the replicon RNA. This is typically done with two defectivehelper RNAs which encode the structural proteins. One helper consists ofa full length infectious clone from which the nonstructural proteingenes and the glycoprotein genes are deleted. This helper retains onlythe terminal nucleotide sequences, the promoter for subgenomic mRNAtranscription and the sequences for the viral nucleocapsid protein. Thesecond helper is identical to the first except that the nucleocapsidgene is deleted and only the glycoprotein genes are retained. The helperRNAs are transcribed in vitro and are co-transfected with replicon RNA.Because the replicon RNA retains the sequences for packaging by thenucleocapsid protein, and because the helpers lack these sequences, onlythe replicon RNA is packaged by the viral structural proteins. Thepackaged replicon particles are released from the host cell and can thenbe purified and inoculated into animals. The packaged replicon particleswill have a tropism similar to the parent virus. The packaged repliconparticles will infect cells and initiate a single round of replication,resulting in the expression of only the virus nonstructural proteins andthe product of the heterologous gene that was cloned in the place of thevirus structural proteins. In the absence of RNA encoding the virusstructural proteins, no progeny virus particles can be produced from thecells infected by packaged replicon particles.

[0022] The Venezuelan equine encephalitis (VEE) virus replicon is agenetically reorganized version of the VEE virus genome in which thegenes encoding the VEE structural proteins are replaced with aheterologous gene of interest. In the present invention, theheterologous genes are the GP, NP, or VP virion proteins from the Ebolavirus. The result is a sell-replicating RNA that can be packaged intoinfectious particles using defective helper RNAs that encode theglycoprotein and capsid proteins of the VEE virus. The replicon and itsuse is further described in U.S. Pat. No. 5,792,462 issued to Johnstonet al. on Aug. 11, 1998.

[0023] Subject. Includes both human, animal, e.g., horse, donkey, pig,mouse, hamster, monkey, chicken, and insect such as mosquito.

[0024] In one embodiment, the present invention relates to DNA fragmentswhich encode any of the Ebola Zaire 1976 (Mayinga isolate) GP, NP, VP24,VP30, VP35, and VP40 proteins. The GP and NP genes of Ebola Zaire werepreviously sequenced by Sanchez et al. (1993, supra) and have beendeposited in GenBank (accession number L11365). A plasmid encoding theVEE replicon vector containing a unique ClaI site downstream from the26S promoter was described previously (Davis, N. L. et al., (1996) J.Virol. 70, 3781-3787; Pushko, P. et al. (1997) Virology 239, 389-401).The Ebola GP and NP genes from the Ebola Zaire 1976 virus were derivedfrom PS64— and PGEM3ZF(−)-based plasmids (Sanchez, A. et al. (1989)Virology 170, 81-91; Sanchez, A. et al. (1993) Virus Res. 29, 215-240).From these plasmids, the BamHI-EcoRI (2.3 kb) and BamHI-KpnI (2.4 kb)fragments containing the NP and GP genes, respectively, were subclonedinto a shuttle vector that had been digested with BamHI and EcoRI (Daviset al. (1996) supra; Grieder, F. B. et al. (1995) Virology 206,994-1006). For cloning of the GP gene, overhanging ends produced by KpnI(in the GP fragment) and EcoRI (in the shuttle vector) were made bluntby incubation with T4 DNA polymerase according to methods known in theart. From the shuttle vector, GP or NP genes were subcloned asClaI-fragments into the ClaI site of the replicon clone, resulting inplasmids encoding the GP or NP genes in place of the VEE structuralprotein genes downstream from the VEE 26S promoter.

[0025] The VP genes of Ebola Zaire were previously sequenced by Sanchezet al. (1993, supra) and have been deposited in GenBank (accessionnumber L11365). The VP genes of Ebola used in the present invention werecloned by reverse transcription of RNA from Ebola-infected Vero E6 cellsand subsequent amplification of viral cDNAs using the polymerase chainreaction. First strand synthesis was primed with oligo dT (LifeTechnologies). Second strand synthesis and subsequent amplification ofviral cDNAs were performed with gene-specific primers (SEQ ID NOS:8-16).The primer sequences were derived from the GenBank deposited sequencesand were designed to contain a ClaI restriction site for cloning theamplified VP genes into the ClaI site of the replicon vector. Theletters and numbers in bold print indicate Ebola gene sequences in theprimers and the corresponding location numbers based on the GenBankdepositied sequences. VP24: (1) forward primer is5′-GGGATCGATCTCCAGACACCAAGCAAGACC-3′ (SEQ ID NO: 8) (10,311-10,331) (2)reverse primer is 5′-GGGATCGATGAGTCAGCATATATGAGTTAGCTC-3′ (SEQ ID NO: 9)(11,122-11,145) VP30: (1) forward primer is5′-CCCATCGATCAGATCTGCGAACCGGTAGAG-3′ SEQ ID NO: 10) (8408-8430) (2)reverse primer is 5′-CCCATCGATGTACCCTCATCAGACCATGAGC-3′ (SEQ ID NO: 11)(9347-9368) VP35: (1) forward primer is5′-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3′ (SEQ ID NO: 12) (3110-3133) (2)reverse primer is 5--CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3′ (SEQ ID NO:13) (4218-4244) VP40: (1) forward primer is5′-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3′ (SEQ ID NO: 14) (4408-4428) (2)reverse primer is 5′-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3′ (SEQ ID NO:15) (5495-5518) VP30 #2: (1) forward primer as for VP30 above (2)reverse primer is 5′-CCCATCGATCTGTTAGGGTTGTATCATACC-3′ (SEQ ID NO: 16)

[0026] The Ebola virus genes cloned into the VEE replicon weresequenced. Changes in the DNA sequence relative to the sequencepublished by Sanchez et al. (1993) are described relative to thenucleotide (nt) sequence number from GenBank (accession number L11365).

[0027] The nucleotide sequence we obtained for Ebola virus GP (SEQ IDNO:1) differed from the GenBank sequence by a transition from A to G atnt 8023. This resulted in a change in the amino acid sequence from Ileto Val at position 662 (SEQ ID NO: 17).

[0028] The nucleotide sequence we obtained for Ebola virus NP (SEQ IDNO:2) differed from the GenBank sequence at the following 4 positions:insertion of a C residue between nt 973 and 974, deletion of a G residueat nt 979, transition from C to T at nt 1307, and a transversion from Ato C at nt 2745. These changes resulted in a change in the proteinsequence from Arg to Glu at position 170 and a change from Leu to Phe atposition 280 (SEQ ID NO: 18).

[0029] The Ebola virus VP24 nucleotide sequence (SEQ ID NO:3) differedfrom the GenBank sequence at 6 positions, resulting in 3 nonconservativechanges in the amino acid sequence. The changes in the DNA sequence ofVP24 consisted of a transversion from G to C at nt 10795, a transversionfrom C to G at nt 10796, a transversion from T to A at nt 10846, atransversion from A to T at nt 10847, a transversion from C to G at nt11040, and a transversion from C to G at nt 11041. The changes in theamino acid sequence of VP24 consisted of a Cys to Ser change at position151, a Leu to His change at position 168, and a Pro to Gly change atposition 233 (SEQ ID NO: 19).

[0030] Two different sequences for the Ebola virus VP30 gene, VP30 andVP30#2 (SEQ ID NOS: 4 and 7) are included. Both of these sequencesdiffer from the GenBank sequence by the insertion of an A residue in theupstream noncoding sequence between nt 8469 and 8470 and an insertion ofa T residue between nt 9275 and 9276 that results in a change in theopen reading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS: 20and 23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 nucleic acid in SEQ ID NO:7 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO: 4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO: 23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We are currently examining theability of the new VEE replicon construct, which we believe contains theauthentic C-terminus of VP30 (VP30#2, SEQ ID NO: 23), to protect miceagainst a lethal Ebola challenge.

[0031] The nucleotide sequence for Ebola virus VP35 (SEQ ID NO:5)differed from the GenBank sequence by a transition from T to C at nt4006, a transition from T to C at nt 4025, and an insertion of a Tresidue between nt 4102 and 4103. These sequence changes resulted in achange from a Ser to a Pro at position 293 and a change from Phe to Serat position 299 (SEQ ID NO: 21). The insertion of the T residue resultedin a change in the open reading frame of VP35 from that previouslyreported by Sanchez et al. (1993) following amino acid number 324. As aresult, Ebola virus VP35 encodes a protein of 340 amino acids, whereamino acids 325 to 340 (SEQ ID NO: 21) differ from and replace theC-terminal 27 amino acids of the previously published sequence.

[0032] Sequencing of VP30 and VP35 was also performed on RT/PCR productsfrom RNA derived from cells that were infected with Ebola virus 1976,Ebola virus 1995 or the mouse-adapted Ebola virus. The changes notedabove for the Vrep constructs were also found in these Ebola viruses.Thus, we believe that these changes are real events and not artifacts ofcloning.

[0033] The Ebola virus VP40 nucleotide sequence (SEQ ID NO:6) differedfrom the GenBank sequence by a transversion from a C to G at nt 4451 anda transition from a G to A at nt 5081. These sequence changes did notalter the protein sequence of VP40 (SEQ ID NO: 22) from that of thepublished sequence.

[0034] DNA or polynucleotide sequences to which the invention alsorelates include sequences of at least about 6 nucleotides, preferably atleast about 8 nucleotides, more preferably at least about 10-12nucleotides, most preferably at least about 15-20 nucleotidescorresponding, i.e., homologous to or complementary to, a region of theEbola nucleotide sequences described above. Preferably, the sequence ofthe region from which the polynucleotide is derived is homologous to orcomplementary to a sequence which is unique to the Ebola genes. Whetheror not a sequence is unique to the Ebola gene can be determined bytechniques known to those of skill in the art. For example, the sequencecan be compared to sequences in databanks, e.g., GenBank and compared byDNA:DNA hybridization. Regions from which typical DNA sequences may bederived include but are not limited to, for example, regions encodingspecific epitopes, as well as non-transcribed and/or non-translatedregions.

[0035] The derived polynucleotide is not necessarily physically derivedfrom the nucleotide sequences shown in SEQ ID NO:1-7, but may begenerated in any manner, including for example, chemical synthesis orDNA replication or reverse transcription or transcription, which arebased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. In addition,combinations of regions corresponding to that of the designated sequencemay be modified in ways known in the art to be consistent with anintended use. The sequences of the present invention can be used indiagnostic assays such as hybridization assays and polymerase chainreaction assays, for example, for the discovery of other Ebolasequences.

[0036] In another embodiment, the present invention relates to arecombinant DNA molecule that includes a vector and a DNA sequence asdescribed above. The vector can take the form of a plasmid, a eukaryoticexpression vector such as pcDNA3.1, pRcCMV2, pZeoSV2, or pCDM8, whichare available from Invitrogen, or a virus vector such as baculovirusvectors, retrovirus vectors or adenovirus vectors, alphavirus vectors,and others known in the art.

[0037] In a further embodiment, the present invention relates to hostcells stably transformed or transfected with the above-describedrecombinant DNA constructs. The host cell can be prokaryotic (forexample, bacterial), lower eukaryotic (for example, yeast or insect) orhigher eukaryotic (for example, all mammals, including but not limitedto mouse and human). Both prokaryotic and eukaryotic host cells may beused for expression of the desired coding sequences when appropriatecontrol sequences which are compatible with the designated host areused.

[0038] Among prokaryotic hosts, E. coli is the most frequently used hostcell for expression. General control sequences for prokaryotes includepromoters and ribosome binding sites. Transfer vectors compatible withprokaryotic hosts are commonly derived from a plasmid containing genesconferring ampicillin and tetracycline resistance (for example, pBR322)or from the various pUC vectors, which also contain sequences conferringantibiotic resistance. These antibiotic resistance genes may be used toobtain successful transformants by selection on medium containing theappropriate antibiotics. Please see e.g., Maniatis, Fitsch and Sambrook,Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes Iand II (D. N. Glover ed. 1985) for general cloning methods. The DNAsequence can be present in the vector operably linked to sequencesencoding an IgG molecule, an adjuvant, a carrier, or an agent for aid inpurification of Ebola proteins, such as glutathione S-transferase.

[0039] In addition, the Ebola virus gene products can also be expressedin eukaryotic host cells such as yeast cells and mammalian cells.Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichiapastoris are the most commonly used yeast hosts. Control sequences foryeast vectors are known in the art. Mammalian cell lines available ashosts for expression of cloned genes are known in the art and includemany immortalized cell lines available from the American Type CultureCollection (ATCC), such as CHO cells, Vero cells, baby hamster kidney(BHK) cells and COS cells, to name a few. Suitable promoters are alsoknown in the art and include viral promoters such as that from SV40,Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus(BPV), and cytomegalovirus (CMV). Mammalian cells may also requireterminator sequences, poly A addition sequences, enhancer sequenceswhich increase expression, or sequences which cause amplification of thegene. These sequences are known in the art.

[0040] The transformed or transfected host cells can be used as a sourceof DNA sequences described above. When the recombinant molecule takesthe form of an expression system, the transformed or transfected cellscan be used as a source of the protein described below.

[0041] In another embodiment, the present invention relates to Ebolavirion proteins such as GP having an amino acid sequence correspondingto SEQ ID NO:17 encompassing 676 amino acids, NP, having an amino acidsequence corresponding to SEQ ID NO:18 encompassing 739 amino acids,VP24, having an amino acid sequence corresponding to SEQ ID NO:19encompassing 251 amino acids, VP30, having an amino acid sequencecorresponding SEQ ID NO:20 encompassing 324 amino acids, VP35, having anamino acid sequence corresponding to SEQ ID NO:21 encompassing 340 aminoacids, and VP40, having an amino acid sequence corresponding to SEQ IDNO:22, encompassing 326 amino acids, and VP30#2, having an amino acidsequence corresponding to SEQ ID NO:23 encompassing 288 amino acids, orany allelic variation of the amino acid sequences. By allelic variationis meant a natural or synthetic change in one or more amino acids whichoccurs between different serotypes or strains of Ebola virus and doesnot affect the antigenic properties of the protein. There are differentstrains of Ebola (Zaire 1976, Zaire 1995, Reston, Sudan, and IvoryCoast). The NP and VP genes of these different viruses have not beensequenced. It would be expected that these proteins would have homologyamong different strains and that vaccination against one Ebola virusstrain might afford cross protection to other Ebola virus strains.

[0042] A polypeptide or amino acid sequence derived from any of theamino acid sequences in SEQ ID NO:17, 18, 19, 20, 21, 22, and 23 refersto a polypeptide having an amino acid sequence identical to that of apolypeptide encoded in the sequence, or a portion thereof wherein theportion consists of at least 2-5 amino acids, preferably at least 8-10amino acids, and more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.

[0043] A recombinant or derived polypeptide is not necessarilytranslated from a designated nucleic acid sequence, or the DNA sequencefound in GenBank accession number L11365. It may be generated in anymanner, including for example, chemical synthesis, or expression from arecombinant expression system.

[0044] When the DNA or RNA sequences described above are in a repliconexpression system, such as the VEE replicon described above, theproteins can be expressed in vivo. The DNA sequence for any of the GP,NP, VP24, VP30, VP35, and VP40 virion proteins can be cloned into themultiple cloning site of a replicon such that transcription of the RNAfrom the replicon yields an infectious RNA encoding the Ebola protein orproteins of interest (see FIG. 2A, 2B and 2C). The replicon constructsinclude Ebola virus GP (SEQ ID NO:1) cloned into a VEE replicon(VRepEboGP), Ebola virus NP (SEQ ID NO:2) cloned into a VEE replicon(VRepEboNP), Ebola virus VP24 (SEQ ID NO:3) cloned into a VEE replicon(VRepEboVP24), Ebola virus VP30 (SEQ ID NO:4) or VP30#2 (SEQ ID NO:7)cloned into a VEE replicon (VRepEboVP30 or VRepEboVP30(#2)), Ebola virusVP35 (SEQ ID NO:5) cloned into a VEE replicon (VRepEboVP35), and Ebolavirus VP40 (SEQ ID NO:6) cloned into a VEE replicon (VRepEboVP40). Thereplicon DNA or RNA can be used as a vaccine for inducing protectionagainst infection with Ebola. Use of helper RNAs containing sequencesnecessary for packaging of the viral replicon transcripts will result inthe production of virus-like particles containing replicon RNAs (FIG.3). These packaged replicons will infect host cells and initiate asingle round of replication resulting in the expression of the Ebolaproteins in said infected cells. The packaged replicon constructs (i.e.VEE virus replicon particles, VRP) include those that express' Ebolavirus GP (EboGPVRP), Ebola virus NP (EboNPVRP), Ebola virus VP24(EboVP24VRP), Ebola virus VP30 (EboVP30VRP or EboVP30VRP(#2)), Ebolavirus VP35 (EboVP35VRP), and Ebola virus VP40 (EboVP40VRP)′.

[0045] In another embodiment, the present invention relates to RNAmolecules resulting from the transcription of the constructs describedabove. The RNA molecules can be prepared by in vitro transcription usingmethods known in the art and described in the Examples below.Alternatively, the RNA molecules can be produced by transcription of theconstructs in vivo, and isolating the RNA. These and other methods forobtaining RNA transcripts of the constructs are known in the art. Pleasesee Current Protocols in Molecular Biology. Frederick M. Ausubel et al.(eds.), John Wiley and Sons, Inc. The RNA molecules can be used, forexample, as a direct RNA vaccine, or to transfect cells along with RNAfrom helper plasmids, one of which expresses VEE glycoproteins and theother VEE capsid proteins, as described above, in order to obtainreplicon particles.

[0046] In a further embodiment, the present invention relates to amethod of producing the recombinant or fusion protein which includesculturing the above-described host cells under conditions such that theDNA fragment is expressed and the recombinant or fusion protein isproduced thereby. The recombinant or fusion protein can then be isolatedusing methodology well known in the art. The recombinant or fusionprotein can be used as a vaccine for immunity against infection withEbola or as a diagnostic tool for detection of Ebola infection.

[0047] In another embodiment, the present invention relates toantibodies specific for the above-described recombinant proteins (orpolypeptides). For instance, an antibody can be raised against a peptidehaving the amino acid sequence of any of SEQ ID NO:17-25, or against aportion thereof of at least 10 amino acids, preferably, 11-15 aminoacids. Persons with ordinary skill in the art using standard methodologycan raise monoclonal and polyclonal antibodies to the protein (orpolypeptide) of the present invention, or a unique portion thereof.Materials and methods for producing antibodies are well known in the art(see for example Goding, In Monoclonal Antibodies: Principles andPractice, Chapter 4, 1986).

[0048] In a further embodiment, the present invention relates to amethod of detecting the presence of antibodies against Ebola virus in asample. Using standard methodology well known in the art, a diagnosticassay can be constructed by coating on a surface (i.e. a solid supportfor example, a microtitration plate, a membrane (e.g. nitrocellulosemembrane) or a dipstick), all or a unique portion of any of the Ebolaproteins described above or any combination thereof, and contacting itwith the serum of a person or animal suspected of having Ebola. Thepresence of a resulting complex formed between the Ebola protein(s) andserum antibodies specific therefor can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of Ebola infection and for determining the degree to which anindividual has developed virus-specific Abs after administration of avaccine.

[0049] In yet another embodiment, the present invention relates to amethod for detecting the presence of Ebola virion proteins in a sample.Antibodies against GP, NP, and the VP proteins could be used fordiagnostic assays. Using standard methodology well known in the art, adiagnostics assay can be constructed by coating on a surface (i.e. asolid support, for example, a microtitration plate or a membrane (e.g.nitrocellulose membrane)), antibodies specific for any of the Ebolaproteins described above, and contacting it with serum or a tissuesample of a person suspected of having Ebola infection. The presence ofa resulting complex formed between the protein or proteins in the serumand antibodies specific therefor can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of Ebola virus infection.

[0050] In another embodiment, the present invention relates to adiagnostic kit which contains any combination of the Ebola proteinsdescribed above and ancillary reagents that are well known in the artand that are suitable for use in detecting the presence of antibodies toEbola in serum or a tissue sample. Tissue samples contemplated can befrom monkeys, humans, or other mammals.

[0051] In yet another embodiment, the present invention relates to DNAor nucleotide sequences for use in detecting the presence of Ebola virususing the reverse transcription-polymerase chain reaction (RT-PCR). TheDNA sequence of the present invention can be used to design primerswhich specifically bind to the viral RNA for the purpose of detectingthe presence of Ebola virus or for measuring the amount of Ebola virusin a sample. The primers can be any length ranging from 7 to 400nucleotides, preferably at least 10 to 15 nucleotides, or morepreferably 18 to 40 nucleotides. Reagents and controls necessary for PCRreactions are well known in the art. The amplified products can then beanalyzed for the presence of viral sequences, for example by gelfractionation, with or without hybridization, by radiochemistry, andimmunochemistry techniques.

[0052] In yet another embodiment, the present invention relates to adiagnostic kit which contains PCR primers specific for Ebola virus andancillary reagents for use in detecting the presence or absence of Ebolain a sample using PCR. Samples contemplated can be obtained from human,animal, e.g., horse, donkey, pig, mouse, hamster, monkey, or othermammals, birds, and insects, such as mosquitoes.

[0053] In another embodiment, the present invention relates to an Ebolavaccine comprising VRPs that express one or more of the Ebola proteinsdescribed above. The vaccine is administered to a subject wherein thereplicon is able to initiate one round of replication producing theEbola proteins to which a protective immune response is initiated insaid subject.

[0054] It is likely that the protection afforded by these genes is dueto both the humoral (antibodies (Abs)) and cellular (cytotoxic T cells(CTLs)) arms of the immune system. Protective immunity induced to aspecific protein may comprise humoral immunity, cellular immunity, orboth. The only Ebola virus protein known to be on the outside of thevirion is the GP. The presence of GP on the virion surface makes it alikely target for GP-specific Abs that may bind either extracellularvirions or infected cells expressing GP on their surfaces. Serumtransfer studies in this invention demonstrate that Abs that recognizeGP protect mice against lethal Ebola virus challenge.

[0055] In contrast, transfer of Abs specific for NP, VP24, VP30, VP35,or VP40 did not protect mice against lethal Ebola challenge. This data,together with the fact that these are internal virion proteins that arenot readily accessible to Abs on either extracellular virions or thesurface of infected cells, suggest that the protection induced in miceby these proteins is mediated by CTLs.

[0056] CTLs can bind to and lyse virally infected cells. This processbegins when the proteins produced by cells are routinely digested intopeptides. Some of these peptides are bound by the class I or class IImolecules of the major histocompatability complex (MHC), which are thentransported to the cell surface. During virus infections, viral proteinsproduced within infected cells also undergo this process. CTLs that havereceptors that bind to both a specific peptide and the MHC moleculeholding the peptide lyse the peptide-bearing cell, thereby limitingvirus replication. Thus, CTLs are characterized as being specific for aparticular peptide and restricted to a class I or class II MHC molecule.

[0057] CTLs may be induced against any of the Ebola virus proteins, asall of the viral proteins are produced and digested within the infectedcell. Thus, protection to Ebola virus could involve CTLs against GP, NP,VP24, VP30, VP35, and/or VP40. It is especially noteworthy that the VPproteins varied in their protective efficacy when tested in geneticallyinbred mice that differ at the MHC locus. This, together with theinability to demonstrate a role for Abs in protection induced by the VPproteins, strongly supports a role for CTLs. These data also suggestthat an eventual vaccine candidate may include several Ebola virusproteins, or several CTL epitopes, capable of inducing broad protectionin outbred populations (e.g. people). We have identified two sequencesrecognized by CTLs. They are Ebola virus NP SEQ ID NO:24 and Ebola virusVP24 SEQ ID NO:25. Testing is in progress to identify the role of CTLsin protection induced by each of these Ebola virus proteins and todefine the minimal sequence requirements for the protective response.The CTL assay is well known in the art.

[0058] An eventual vaccine candidate might comprise these CTL sequencesand others. These might be delivered as synthetic peptides, or as fusionproteins, alone or co-administered with cytokines and/or adjuvants orcarriers safe for human use, e.g. aluminum hydroxide, to increaseimmunogenicity. In addition, sequences such as ubiquitin can be added toincrease antigen processing for more effective CTL responses.

[0059] In yet another embodiment, the present invention relates to amethod for providing immunity against Ebola virus, said methodcomprising administering one or more VRPs expressing any combination ofthe GP, NP, VP24, VP30 or VP30#2, VP35 and VP40 Ebola proteins to asubject such that a protective immune reaction is generated.

[0060] Vaccine formulations of the present invention comprise animmunogenic amount of a VRP, such as for example EboVP24VRP describedabove, or, for a multivalent vaccine, a combination of replicons, in apharmaceutically acceptable carrier. An “immunogenic amount” is anamount of the VRP(s) sufficient to evoke an immune response in thesubject to which the vaccine is administered. An amount of from about104-10⁸ focus-forming units per dose is suitable, depending upon the ageand species of the subject being treated. The subject may be inoculated2-3 times. Exemplary pharmaceutically acceptable carriers include, butare not limited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution.

[0061] Administration of the VRPs disclosed herein may be carried out byany suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), in ovoinjection of birds, orally, or by topical application of the virus(typically carried in a pharmaceutical formulation) to an airwaysurface. Topical application of the virus to an airway surface can becarried out by intranasal administration (e.g., by use of dropper, swab,or inhaler which deposits a pharmaceutical formulation intranasally).Topical application of the virus to an airway surface can also becarried out by inhalation administration, such as by creating respirableparticles of a pharmaceutical formulation (including both solidparticles and liquid particles) containing the replicon as an aerosolsuspension, and then causing the subject to inhale the respirableparticles. Methods and apparatus for administering respirable particlesof pharmaceutical formulations are well known, and any conventionaltechnique can be employed. Oral administration may be in the form of aningestable liquid or solid formulation.

[0062] When the replicon RNA or DNA is used as a vaccine, the repliconRNA or DNA can be administered directly using techniques such asdelivery on gold beads (gene gun), delivery by liposomes, or directinjection, among other methods known to people in the art. Any one ormore DNA constructs or replicating RNA described above can be use in anycombination effective to elicit an immunogenic response in a subject.Generally, the nucleic acid vaccine administered may be in an amount ofabout 1-5 ug of nucleic acid per dose and will depend on the subject tobe treated, capacity of the subject's immune system to develop thedesired immune response, and the degree of protection desired. Preciseamounts of the vaccine to be administered may depend on the judgement ofthe practitioner and may be peculiar to each subject and antigen.

[0063] The vaccine may be given in a single dose schedule, or preferablya multiple dose schedule in which a primary course of vaccination may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the immuneresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitableimmunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or otherschedules sufficient to elicit the desired immune responses expected toconfer protective immunity, or reduce disease symptoms, or reduceseverity of disease.

[0064] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors and thought to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be % made in the specific embodiments which are disclosedand still obtain a like or similar result without departing from thespirit and scope of the invention.

[0065] The following MATERIALS AND METHODS were used in the examplesthat follow.

[0066] Cells Lines and Viruses

[0067] BHK (ATCC CCL 10), Vero 76 (ATCC CRL 1587), and 11 Vero E6 (ATCCCRL 1586) cell lines were maintained in minimal essential medium withEarle's salts, 5-10% fetal bovine serum, and 50 μg/mL gentamicinsulfate. For CTL assays, EL4 (ATCC TIB39), L5178Y (ATCC CRL 1723) andP815 (ATCC TIB64) were maintained in Dulbecco's minimal essential mediumsupplemented with 5-10% fetal bovine serum and antibiotics.

[0068] A stock of the Zaire strain of Ebola virus originally isolatedfrom a patient in the 1976 outbreak (Mayinga) and passagedintracerebrally 3 times in suckling mice and 2 times in Vero cells wasadapted to adult mice through serial passage in progressively oldersuckling mice (Bray et al., (1998) J. Infect. Dis. 178, 651-661). Aplaque-purified ninth-mouse-passage isolate which was uniformly lethalfor adult mice (“mouse-adapted virus”) was propagated in Vero E6 cells,aliquotted, and used in all mouse challenge experiments andneutralization assays.

[0069] A stock of the Zaire strain of Ebola 1976 virus was passagedspleen to spleen in strain 13 guinea pigs four times. This guineapig-adapted strain was used to challenge guinea pigs.

[0070] Construction and Packaging of Recombinant VEE Virus Replicons(VRPs)

[0071] Replicon RNAs were packaged into VRPs as described (Pushko etal., 1997, supra). Briefly, capped replicon RNAs were produced in vitroby T7 run-off transcription of NotI-digested plasmid templates using theRiboMAX T7 RNA polymerase kit (Promega). BHK cells were co-transfectedwith the replicon RNAs and the 2 helper RNAs expressing the structuralproteins of the VEE virus. The cell culture supernatants were harvestedapproximately 30 hours after transfection and the replicon particleswere concentrated and purified by centrifugation through a 20% sucrosecushion. The pellets containing the packaged replicon particles weresuspended in PBS and the titers were determined by infecting Vero cellswith serial dilutions of the replicon particles and enumerating theinfected cells by indirect immunofluorescence with antibodies specificfor the Ebola proteins.

[0072] Immunoprecipitation of Ebola Virus Proteins Expressed from VEEVirus Replicons

[0073] BHK cells were transfected with either the Ebola virus GP, NP,VP24, VP30, VP35, or VP40 replicon RNAS. At 24 h post-transfection, theculture medium was replaced with minimal medium lacking cysteine andmethionine, and proteins were labeled for 1 h with ³⁵S-labeledmethionine and cysteine. Cell lysates or supernatants (supe) werecollected and immunoprecipitated with polyclonal rabbit anti-Ebola virusserum bound to protein A beads. ³⁵S-labeled Ebola virus structuralproteins from virions grown in Vero E6 cells were alsoimmunoprecipitated as a control for each of the virion proteins.Immunoprecipitated proteins were resolved by electrophoresis on an 11%SDS-polyacrylamide gel and were visualized by autoradiography.

[0074] Vaccination of Mice with VEE Virus Replicons

[0075] Groups of 10 BALB/c or C57BL/6 mice per experiment weresubcutaneously injected at the base of the neck with 2×10⁶ focus-formingunits of VRPs encoding the Ebola virus genes. As controls, mice werealso injected with either a control VRP encoding the Lassa nucleoprotein(NP) or with PBS. For booster inoculations, animals received identicalinjections at 1 month intervals. Data are recorded as the combinedresults of 2 or 3 separate experiments.

[0076] Ebola Infection of Mice

[0077] One month after the final booster inoculation, mice weretransferred to a BSL-4 containment area and challenged byintraperitoneal (ip) inoculation of 10 plaque-forming units (pfu) ofmouse-adapted Ebola virus (approximately 300 times the dose lethal for50% of adult mice). The mice were observed daily, and morbidity andmortality were recorded. Animals surviving at day 21 post-infection wereinjected again with the same dose of virus and observed for another 21days.

[0078] In some experiments, 4 or 5 mice from vaccinated and controlgroups were anesthetized and exsanguinated on day 4 (BALB/c mice) or day5 (C57BL/6 mice) following the initial viral challenge. The viral titersin individual sera were determined by plaque assay.

[0079] Passive Transfer of Immune Sera to Naive Mice.

[0080] Donor sera were obtained 28 days after the third inoculation with2×10⁶ focus-forming units of VRPs encoding the indicated Ebola virusgene, the control Lassa NP gene, or from unvaccinated control mice. OnemL of pooled donor sera was administered intraperitoneally (ip) tonaive, syngeneic mice 24 h prior to intraperitoneal challenge with 10pfu of mouse-adapted Ebola virus.

[0081] Vaccination and Challenae of Guinea Pigs.

[0082] EboGPVRP or EboNPVRP (1×10⁷ focus-forming units in 0.5 ml PBS)were administered subcutaneously to inbred strain 2 or strain 13 guineapigs (300-400 g). Groups of five guinea pigs were inoculated on days 0and 28 at one (strain 2) or two (strain 13) dorsal sites. Strain 13guinea pigs were also boosted on day 126. One group of Strain 13 guineapigs was vaccinated with both the GP and NP constructs. Blood sampleswere obtained after vaccination and after viral challenge. Guinea pigswere challenged on day 56 (strain 2) or day 160 (strain 13) bysubcutaneous administration of 1000 LD₅₀ (1×10⁴ PFU) of guineapig-adapted Ebola virus. Animals were observed daily for 60 days, andmorbidity (determined as changes in behavior, appearance, and weight)and survival were recorded. Blood samples were taken on the daysindicated after challenge and viremia levels were determined by plaqueassay.

[0083] Virus titration and neutralization assay. Viral stocks wereserially diluted in growth medium, adsorbed onto confluent Vero E6 cellsin 6- or 12-well dishes, incubated for 1 hour at 37° C., and coveredwith an agarose overlay (Moe, J. et al. (1981) J. Clin. Microbiol.13:791-793). A second overlay containing 5% neutral red solution in PBSor agarose was added 6 days later, and plaques were counted thefollowing day. Pooled pre-challenge serum samples from some of theimmunized groups were tested for the presence of Ebola-neutralizingantibodies by plaque reduction neutralization assay. Aliquots of Ebolavirus in growth medium were mixed with serial dilutions of test serum,or with normal serum, or medium only, incubated at 37° C. for 1 h, andused to infect Vero E6 cells. Plaques were counted 1 week later.

[0084] Cytotoxic T cell assays. BALB/c and C57BL/6 mice were inoculatedwith VRPs encoding Ebola virus NP or VP24 or the control Lassa NPprotein. Mice were euthanized at various times after the lastinoculation and their spleens removed. The spleens were gently rupturedto generate single cell suspensions. Spleen cells (1×10⁶/ml) werecultured in vitro for 2 days in the presence of 10-25 μM of peptidessynthesized from Ebola virus NP or VP24 amino acid sequences, and thenfor an additional 5 days in the presence of peptide and 10% supernatantfrom concanavalin A-stimulated syngeneic spleen cells. Syntheticpeptides were made from Ebola virus amino acid sequences 1 predicted bya computer algorithm (HLA Peptide Binding Predictions, Parker, K. C., etal. (1994) J. Immunol. 152:163) to have a likelihood of meeting the MHCclass I binding requirements of the BALB/c (H-2^(d)) and C57BL/6(H-2^(b)) haplotypes. Only 2 of 8 peptides predicted by the algorithmand tested to date have been identified as containing CTL epitopes.After in vitro restimulation, the spleen cells were tested in a standard⁵¹chromium-release assay well known in the art (see, for example, Hartet al. (1991) Proc. Natl. Acad. Sci. USA 88: 9449-9452). Percentspecific lysis of peptide-coated, MHC-matched or mismatched target cellswas calculated as:$\frac{{{Experimental}\quad {cpm}} - {{Spontaneous}\quad {cpm} \times 100}}{{{Maximum}\quad {cpm}} - {{Spontaneous}\quad {cpm}}}$

[0085] Spontaneous cpm are the number of counts released from targetcells incubated in medium. Maximum cpm are obtained by lysing targetcells with 1% Triton X-100. Experimental cpm are the counts from wellsin which target cells are incubated with varying numbers of effector(CTL) cells. Target cells tested were L5178Y lymphoma or P815mastocytoma cells (MHC matched to the H2^(d) BALB/c mice and EL4lymphoma cells (MHC matched to the H2^(b) C57BL/6 mice). Theeffector:target (E:T) ratios tested were 25:1, 12:1, 6:1 and 3:1.

EXAMPLE 1

[0086] Survival Of Mice Inoculated With VRPs Encoding Ebola Proteins.Mice were inoculated two or three times at 1 month intervals with 2×10⁶focus-forming units of VRPs encoding individual Ebola virus genes, orLassa virus NP as a control, or with phosphate buffered saline (PBS).Mice were challenged with 10 pfu of mouse-adapted Ebola virus one monthafter the final immunization. The mice were observed daily, andmorbidity and mortality data are shown in Table 1A for BALB/c mice andTable 1B for C57BL/6 mice. The viral titers in individual sera of somemice on day 4 (BALB/c mice) or day 5 (C57BL/6 mice) following theinitial viral challenge were determined by plaque assay. TABLE 1Survival Of Mice Inoculated With VRPs Encoding Ebola Proteins VRP#Injections S/T¹ (%) MDD² V/T³ Viremia⁴ A. BALB/c Mice EboNP 3 30/30(100%) 5/5 5.2 2 19/20 (95%) 7 5/5 4.6 EboGP 3 15/29 (52%) 8 1/5 6.6 214/20 (70%) 7 3/5 3.1 EboVP24 3 27/30 (90%) 8 5/5 5.2 2 19/20 (95%) 64/4 4.8 EboVP30 3 17/20 (85%) 7 5/5 6.2 2 11/20 (55%) 7 5/5 6.5 EboVP353  5/19 (26%) 7 5/5 6.9 2  4/20 (20%) 7 5/5 6.5 EboVP40 3 14/20 (70%) 85/5 4.6 2 17/20 (85%) 7 5/5 5.6 LassaNP 3  0/29 (0%) 7 5/5 8.0 2  0/20(0%) 7 5/5 8.4 none (PBS) 3  1/30 (3%) 6 5/5 8.3 2  O/2O (O%) 6 5/5 8.7B. C57BL/6 Mice EboNP 3 15/20 (75%) 8 5/5 4.1 2  8/10 (80%) 9  ND⁵ NDEboGP 3 19/20 (95%) 10  0/5 — 2 10/10 (100%) — ND ND EboVP24 3  0/20(0%) 7 5/5 8.6 EboVP30 3  2/20 (10%) 8 5/5 7.7 EboVP35 3 14/20 (70%) 85/5 4.5 EboVP40 3  1/20 (5%) 7 4/4 7.8 LassaNP 3  1/20 (5%) 7 4/4 8.6 2 0/10 (0%) 7 ND ND none (PBS) 3  3/20 (15%) 7 5/5 8.6 2  0/10 (0%) 7 NDND

EXAMPLE 2

[0087] VP24-Immunized BALB/c Mice Survive A High-Dose Challenge WithEbola virus.

[0088] BALB/c mice were inoculated two times with 2×10⁶ focus-formingunits of EboVP24VRP. Mice were challenged with either 1×10³ pfu or 1×10pfu of mouse-adapted Ebola virus 1 month after the second inoculation.Morbidity and mortality data for these mice are shown in Table 2. TABLE2 VP24-Immunized BALB/c Mice Survive A High- Dose Challenge With Ebolavirus Replicon Challenge Dose Survivors/Total EboVP24 1 × 10³ pfu 5/5 (3× 10⁴ LD₅₀) EboVP24 1 × 10⁵ pfu 5/5 (3 × 10⁶ LD₅₀) None 1 × 10³ pfu 0/4(3 × 10⁴ LD₅₀) None 1 × 10⁵ pfu 0/3 (3 × 10⁶ LD₅₀)

EXAMPLE 3

[0089] Passive Transfer Of Immune Sera Can Protect Naive Mice From ALethal Challenae Of Ebola Virus. Donor sera were obtained 28 days afterthe third inoculation with 2×10⁶ focus-forming units of VRPs encodingthe indicated Ebola virus gene, the control Lassa NP gene, or fromunvaccinated control mice. One ml of pooled donor sera was administeredintraperitoneally (ip) to naive, syngeneic mice 24 h prior tointraperitoneal challenge with 10 pfu of mouse-adapted Ebola virus.TABLE 3 Passive Transfer of Immune Sera Can Protect Unvaccinated Micefrom a Lethal Challenge of Ebola Virus Survivors/ Mean Day Total ofDeath A. BALB/c Mice Specificity of Donor Sera⁻ Ebola GP 15/20 8 EbolaNP  1/20 7 Ebola VP24  0/20 6 Ebola VP30  0/20 7 Ebola VP35  ND¹ NDEbola VP40  0/20 6 Lassa NP  0/20 7 Normal mouse sera  0/20 6 B. C57BL/6Mice Specificity of Donor Sera⁻ Ebola GP 17/20 7 Ebola NP  0/20 7 EbolaVP24 ND ND Ebola VP30 ND ND Ebola VP35  0/20 7 Ebola VP40 ND ND Lassa NP 0/20 7 Normal mouse sera  0/20 7

EXAMPLE 4

[0090] Immunogenicity and Efficacy of VRepEboGP and VRepEboNP in GuineaPigs.

[0091] EboGPVRP or EboNPVRP (1×10⁷ IU in 0.5 ml PBS) were administeredsubcutaneously to inbred strain 2 or strain 13 guinea pigs (300-400 g).Groups of five guinea pigs were inoculated on days 0 and 28 at one(strain 2) or two (strain 13) dorsal sites. Strain 13 guinea pigs werealso boosted on day 126. One group of Strain 13 guinea pigs wasvaccinated with both the GP and NP constructs. Blood samples wereobtained after vaccination and after viral challenge.

[0092] Sera from vaccinated animals were assayed for antibodies to Ebolaby plaque-reduction neutralization, and ELISA. Vaccination withVRepEboGP or NP induced high titers of antibodies to the Ebola proteins(Table 4) in both guinea pig strains. Neutralizing antibody responseswere only detected in animals vaccinated with the GP construct (Table4).

[0093] Guinea pigs were challenged on day 56 (strain 2) or day 160(strain 13) by subcutaneous administration of 1000 LD₅₀ (10⁴ PFU) ofguinea pig-adapted Ebola virus. Animals were observed daily for 60 days,and morbidity (determined as changes in behavior, appearance, andweight) and survival were recorded. Blood samples were taken on the daysindicated after challenge and viremia levels were determined by plaqueassay. Strain 13 guinea pigs vaccinated with the GP construct, alone orin combination with NP, survived lethal Ebola challenge (Table 4).Likewise, vaccination of strain 2 inbred guinea pigs with the GPconstruct protected 3/5 animals against death from lethal Ebolachallenge, and significantly prolonged the mean day of death (MDD) inone of the two animals that died (Table 4). Vaccination with NP alonedid not protect either guinea pig strain. TABLE 4 Immunogenicity andefficacy of VRepEboGP and VRepEboNP in guinea pigs Survivors/Viremia^(c) VRP ELISA^(a) PRNT₅₀ total (MDD^(b)) d7 d14 A. Strain 2guinea pigs GP 4.1 30 3/5 (13 + 2.8) 2.3   1.8 NP 3.9 <10 0/5 (9.2 +1.1) 3.0 — Mock <1.5 <10 0/5 (8.8 + 0.5) 3.9 — B. Strain 13 guinea pigsGP 4.0 140 5/5 <2.0 <2.0 GP/NP 3.8 70 5/5 <2.0 <2.0 NP 2.8 <10 1/5(8.3 + 2.2) 4.6 — Lassa NP <1.5 <10 2/5 (8.3 + 0.6) 4.8 —

EXAMPLE 5

[0094] Induction of murine CTL responses to Ebola virus NP and Ebolavirus VP24 proteins.

[0095] BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebolavirus NP or VP24. Mice were euthanized at various times after the lastinoculation and their spleens removed. Spleen cells (1×10⁶/ml) werecultured in vitro for 2 days in the presence of 10 to 25 μM of peptides,and then for an additional 5 days in the presence of peptide and 10%supernatant from concanavalin A-stimulated syngeneic spleen cells. 0.34After in vitro restimulation, the spleen cells were tested in a standard⁵¹chromium-release assay. Percent specific lysis of peptide-coated,MHC-matched or mismatched target cells was calculated as:$\frac{{{Experimental}\quad {cpm}} - {{Spontaneous}\quad {cpm} \times 100}}{{{Maximum}\quad {cpm}} - {{Spontaneous}\quad {cpm}}}$

[0096] In the experiments shown, spontaneous release did not exceed 15%.TABLE 5 Induction of murine CTL responses to Ebola virus NP and Ebolavirus VP24 proteins. % Specific Lysis E:T ratio Mice, VRP¹ Peptide²Cell³ 25 BALB/c, VP24 None P815 55 BALB/c, VP24 SEQ ID NO: 25 P815 93C57BL/6, EboNP None EL4  2 C57BL/6, EboNP⁴ SEQ ID NO: 24 EL4 70 C57BL/6,EboNP Lassa NP EL4  2 C57BL/6, LassaNP None L5178Y  1 C57BL/6, LassaNPSEQ ID NO: 24 L5178Y  0 C57BL/6, LassaNP None EL4  2 C57BL/6, LassaNPSEQ ID NO: 24 EL4  6

Results and Discussion

[0097] Ebola Zaire 1976 (Mayinga) virus causes acute hemorrhagic fevercharacterized by high mortality. There are no current vaccines oreffective therapeutic measures to protect individuals who are exposed tothis virus. In addition, it is not known which genes are essential forevoking protective immunity and should therefore be included in avaccine designed for human use. In this study, the GP, NP, VP24, VP30,VP35, and VP40 virion protein genes of the Ebola Zaire 1976 (Mayinga)virus were cloned and inserted into a Venezuelan equine encephalitis(VEE) virus replicon vector (VRep) as shown in FIG. 2A and 2B. TheseVReps were packaged as VEE replicon particles (VRPs) using the VEE virusstructural proteins provided as helper RNAs, as shown in FIG. 3. Thisenables expression of the Ebola virus proteins in host cells. The Ebolavirus proteins produced from these constructs were characterized invitro and were shown to react with polyclonal rabbit anti-Ebola virusantibodies bound to Protein A beads following SDS gel electrophoresis ofimmunoprecipitated proteins (FIG. 4).

[0098] The Ebola virus genes were sequenced from the VEE replicon clonesand are listed here as SEQ ID NO:1 (GP), 2 (NP), 3 (VP24), 4 (VP30), 5(VP35), 6 (VP40), and 7 (VP30#2) as described below. The correspondingamino acid sequences of the Ebola proteins expressed from thesereplicons are listed as SEQ ID NO: 17, 18, 19, 20, 21, 22, and 23,respectively. Changes in the DNA sequence relative to the sequencepublished by Sanchez et al. (1993) are described relative to thenucleotide (nt) sequence number from GenBank (accession number L11365).

[0099] The sequence we obtained for Ebola virus GP (SEQ ID NO:1)differed from the GenBank sequence by a transition from A to G at nt8023. This resulted in a change in the amino acid sequence from Ile toVal at position 662 (SEQ ID NO: 17).

[0100] The DNA sequence we obtained for Ebola virus NP (SEQ ID NO:2)differed from the GenBank sequence at the following 4 positions:insertion of a C residue between nt 973 and 974, deletion of a G residueat nt 979, transition from C to T at nt 1307, and a transversion from Ato C at nt 2745. These changes resulted in a change in the proteinsequence from Arg to Glu at position 170 and a change from Leu to Phe atposition 280 (SEQ ID NO: 18).

[0101] The Ebola virus VP24 (SEQ ID NO:3) gene differed from the GenBanksequence at 6 positions, resulting in 3 nonconservative changes in theamino acid sequence. The changes in the DNA sequence of VP24 consistedof a transversion from G to C at nt 10795, a transversion from C to G atnt 10796, a transversion from T to A at nt 10846, a transversion from Ato T at nt 10847, a transversion from C to G at nt 11040, and atransversion from C to G at nt 11041. The changes in the amino acidsequence of VP24 consisted of a Cys to Ser change at position 151, a Leuto His change at position 168, and a Pro to Gly change at position 233(SEQ ID NO: 19).

[0102] We have included 2 different sequences for the Ebola virus VP30gene (SEQ ID NOS:4 and SEQ ID NO:7). Both of these sequences differ fromthe GenBank sequence by the insertion of an A residue in the upstreamnoncoding sequence between nt 8469 and 8470 and an insertion of a Tresidue between nt 9275 and 9276 that results in a change in the openreading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS:20 andSEQ ID NO:23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 gene in SEQ ID NO:23 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO:4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO:23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We are currently examining theability of the new VEE replicon construct (SEQ ID NO:7), which webelieve contains the authentic C-terminus of VP30 (VP30#2, SEQ IDNO:23), to protect:mice against a lethal Ebola challenge.

[0103] The DNA sequence for Ebola virus VP35 (SEQ ID NO:5′ differed fromthe GenBank sequence by a transition from T to C at nt 4006, atransition from T to C at nt 4025, and an insertion of a T residuebetween nt 4102 and 4103. These sequence changes resulted in a changefrom a Ser to a Pro at position 293 and a change from Phe to Ser atposition 299 (SEQ ID NO:21). The insertion of the T residue resulted ina change in the open reading frame of VP35 from that previously reportedby Sanchez et al. (1993) following amino acid number 324. As a result,Ebo'a virus VP35 encodes for a protein of 340 amino acids, where aminoacids 325 to 340 (SEQ ID NO:21) differ from and replace the C-terminal27 amino acids of the previously published sequence.

[0104] Sequencing of VP30 and VP35 was also performed on RT/PCR productsfrom RNA derived from cells that were infected with Ebola virus 1976,Ebola virus 1995 or the mouse-adapted Ebola virus. The changes notedabove for the VRep constructs were also found in these Ebola viruses.Thus, we believe that these changes are real events and not artifacts ofcloning.

[0105] The Ebola virus VP40 differed from the GenBank sequence by atransversion from a C to G at nt 4451 and a transition from a G to A atnt 5081. These sequence changes did not alter the protein sequence ofVP40 (SEQ ID NO:22) from that of the published sequence.

[0106] To evaluate the protective efficacy of individual Ebola virusproteins and to determine whether the major histocompatibility (MHC)genes influence the immune response to Ebola virus antigens, twoMHC-incompatible strains of mice were vaccinated with VRPs expressing anEbola protein. As controls for these experiments, some mice wereinjected with VRPs expressing the nucleoprotein of Lassa virus or wereinjected with phosphate-buffered saline (PBS). Following Ebola viruschallenge, the mice were monitored for morbidity and mortality, and theresults are shown in Table 1.

[0107] The GP, NP, VP24, VP30, and VP40 proteins of Ebola virusgenerated either full or partial protection in BALB/c mice, and maytherefore be beneficial components of a vaccine designed for human use.Vaccination with VRPs encoding the NP protein afforded the bestprotection. In this case, 100% of the mice were protected after threeinoculations and 95% of the mice were protected after two inoculations.The VRP encoding VP24 also protected 90% to 95% of BALB/c mice againstEbola virus challenge. In separate experiments. (Table 2), two or threeinoculations with VRPs encoding the VP24 protein protected BALB/c micefrom a high dose (1×10⁵ plaque-forming units (3×10⁶ LD50)) ofmouse-adapted Ebola virus.

[0108] Vaccination with VRPs encoding GP protected 52-70% of BALB/cmice. The lack of protection was not due to a failure to respond to theVRP encoding GP, as all mice had detectable Ebola virus-specific serumantibodies after vaccination.

[0109] Some protective efficacy was also observed in BALB/c micevaccinated two or three times with VRPs expressing the VP30 protein (55%and 85%, respectively), or the VP40 protein (70% and 80%, respectively).The VP35 protein was not efficacious in the BALB/c mouse model, as only20% and 26% of the mice were protected after-either two or three doses,respectively.

[0110] Geometric mean titers of viremia were markedly reduced in BALB/cmice vaccinated with VRPs encoding Ebola virus proteins after challengewith Ebola virus, indicating an ability of the induced immune responsesto reduce virus replication (Table 1A). In this study, immune responsesto the GP protein were able to clear the virus to undetectable levelswithin 4 days after challenge in some mice.

[0111] When the same replicons were examined for their ability toprotect C57BL/6 mice from a lethal challenge of Ebola virus, only theGP, NP, and VP35 proteins were efficacious (Table 1B). The bestprotection, 95% to 100%, was observed in C57BL/6 mice inoculated withVRPs encoding the GP protein. Vaccination with VRPs expressing NPprotected 75% to 80% of the mice from lethal disease. In contrast towhat was observed in the BALB/c mice, the VP35 protein was the only VPprotein able to significantly protect the C57BL/6 mice. In this case, 3inoculations with VRPs encoding VP35 protected 70% of the mice fromEbola virus challenge. The reason behind the differences in protectionin the two mouse strains is not known but is believed to be due to theability of the immunogens to sufficiently stimulate the cellular immunesystem. As with the BALB/c mice, the effects of the induced immuneresponses were also observed in reduced viremias and, occasionally, in aprolonged time to death of C57BL/6 mice.

[0112] VRPs expressing Ebola virus GP or NP were also evaluated forprotective efficacy in a guinea pig model. Sera from vaccinated animalswere assayed for antibodies to Ebola by western blotting, IFA,plaque-reduction neutralization, and ELISA. Vaccination with either VRP(GP or NP) induced high titers of antibodies to the Ebola proteins(Table 4) in both guinea pig strains. Neutralizing antibody responseswere only detected in animals vaccinated with the VRP expressing GP(Table 4).

[0113] Vaccination of strain 2 inbred guinea pigs with the GP constructprotected 3/5 animals against death from lethal Ebola challenge, andsignificantly prolonged the mean day of death in one of the two animalsthat died (Table 4). All of the strain 13 guinea pigs vaccinated withthe GP construct, alone or in combination with NP, survived lethal Ebolachallenge (Table 4). Vaccination with NP alone did not protect eitherguinea pig strain from challenge with the guinea pig-adapted Ebolavirus.

[0114] To identify the immune mechanisms that mediate protection againstEbola virus and to determine whether antibodies are sufficient toprotect against lethal disease, passive transfer studies were performed.One mL of immune sera, obtained from mice previously vaccinated with oneof the Ebola virus VRPs, was passively administered to unvaccinated mice24 hours before challenge with a lethal dose of mouse-adapted Ebolavirus. Antibodies to GP, but not to NP or the VP proteins, protectedmice from an Ebola virus challenge (Table 3). Antibodies to GP protected75% of the BALB/c mice and 85% of the C57BL/6 mice from death. When thedonor sera were examined for their ability to neutralize Ebola virus ina plaque-reduction neutralization assay, a 1:20 to 1:40 dilution of theGP-specific antisera reduced the number of viral plaque-forming units byat least 50% (data not shown). In contrast, antisera to the NP and VPproteins did not neutralize Ebola virus at a 1:20 or 1:40 dilution.These results are consistent with the finding that GP is the only viralprotein found on the surface of Ebola virus, and is likely to inducevirus-neutralizing antibodies.

[0115] Since the NP and VP proteins of Ebola virus are internal virionproteins to which antibodies are not sufficient for protection, it islikely that cytotoxic T lymphocytes (CTLs) are also important forprotection against Ebola virus. Initial studies aimed at identifyingcellular immune responses to individual Ebola virus proteins expressedfrom VRPs identified CTL responses to the VP24: and NP proteins (Table5). One CTL epitope that we identified for the Ebola virus NP isrecognized by C57BL/6 (H-2 b) mice, and has an amino acid sequence of,or contained within, the following 11 amino acids: VYQVNNLEEIC (SEQ IDNO:24). Vaccination with EboNPVRP and in vitro restimulation of spleencells with this peptide consistently induces strong CTL responses inC57BL/6 (H-2^(b)) mice. In vivo vaccination to Ebola virus NP isrequired to detect the CTL activity, as evidenced by the failure ofcells from C57BL/6 mice vaccinated with Lassa NP to develop lyticactivity to peptide (SEQ ID NO:24) after in vitro restimulation with it.Specific lysis has been observed using very low effector:target ratios(<2:1). This CTL epitope is H-2^(b) restricted in that it is notrecognized by BALB/c (H-2^(d)) cells treated the same way (data notshown), and H-2^(b) effector cells will not lyse MHC-mismatched targetcells coated with this peptide.

[0116] A CTL epitope in the VP24 protein was also identified. It isrecognized by BALB/c (H-2^(d)) mice, and has an amino acid sequence of,or contained within, the following 23 amino acids:LKFINKLDALLVVNYNGLLSSIF (SEQ ID NO:25). In the data shown in Table 5,high (>90%) specific lysis of P815 target cells coated with this peptidewas observed. The background lysis of cells that were not peptide-coatedwas also high (>50%), which is probably due to the activity of naturalkiller cells. We are planning to repeat this experiment using the L5178Ytarget cells, which are not susceptible to natural killer cells.

[0117] Future studies will focus on determining the fine specificitiesof these CTL responses and the essential amino acids that constitutethese CTL epitopes. Additional studies to identify other CTL epitopes onEbola virus GP, NP, VP24, VP30, VP35, and VP40 will be performed. Toevaluate the role of these CTLs in protection against Ebola virus,lymphocytes will be restimulated in vitro with peptides containing theCTL epitopes, and adoptively transferred into unvaccinated mice prior toEbola virus challenge. In addition, future studies will examine the CTLresponses to the other Ebola virus proteins to better define the rolesof the cell mediated immune responses involved in protection againstEbola virus infection.

1 25 1 2298 DNA Ebola Zaire 1 atcgataagc tcggaattcg agctcgcccggggatcctct 40 agagtcgaca acaacacaat gggcgttaca ggaatattgc 80 agttacctcgtgatcgattc aagaggacat cattctttct 120 ttgggtaatt atccttttcc aaagaacattttccatccca 160 cttggagtca tccacaatag cacattacag gttagtgatg 200tcgacaaact agtttgtcgt gacaaactgt catccacaaa 240 tcaattgaga tcagttggactgaatctcga agggaatgga 280 gtggcaactg acgtgccatc tgcaactaaa agatggggct320 tcaggtccgg tgtcccacca aaggtggtca attatgaagc 360 tggtgaatgggctgaaaact gctacaatct tgaaatcaaa 400 aaacctgacg ggagtgagtg tctaccagcagcgccagacg 440 ggattcgggg cttcccccgg tgccggtatg tgcacaaagt 480atcaggaacg ggaccgtgtg ccggagactt tgccttccat 520 aaagagggtg ctttcttcctgtatgatcga cttgcttcca 560 cagttatcta ccgaggaacg actttcgctg aaggtgtcgt600 tgcatttctg atactgcccc aagctaagaa ggacttcttc 640 agctcacaccccttgagaga gccggtcaat gcaacggagg 680 acccgtctag tggctactat tctaccacaattagatatca 720 ggctaccggt tttggaacca atgagacaga gtacttgttc 760gaggttgaca atttgaccta cgtccaactt gaatcaagat 800 tcacaccaca gtttctgctccagctgaatg agacaatata 840 tacaagtggg aaaaggagca ataccacggg aaaactaatt880 tggaaggtca accccgaaat tgatacaaca atcggggagt 920 gggccttctgggaaactaaa aaaaacctca ctagaaaaat 960 tcgcagtgaa gagttgtctt tcacagttgtatcaaacgga 1000 gccaaaaaca tcagtggtca gagtccggcg cgaacttctt 1040ccgacccagg gaccaacaca acaactgaag accacaaaat 1080 catggcttca gaaaattcctctgcaatggt tcaagtgcac 1120 agtcaaggaa gggaagctgc agtgtcgcat ctaacaaccc1160 ttgccacaat ctccacgagt ccccaatccc tcacaaccaa 1200 accaggtccggacaacagca cccataatac acccgtgtat 1240 aaacttgaca tctctgaggc aactcaagttgaacaagatc 1280 accgcagaac agacaacgac agcacagcct ccgacactcc 1320ctctgccacg accgcagccg gacccccaaa agcagagaac 1360 accaacacga gcaagagcactgacttcctg gaccccgcca 1400 ccacaacaag tccccaaaac cacagcgaga ccgctggcaa1440 caacaacact catcaccaag ataccggaga agagagtgcc 1480 agcagcgggaagctaggctt aattaccaat actattgctg 1520 gagtcgcagg actgatcaca ggcgggagaagaactcgaag 1560 agaagcaatt gtcaatgctc aacccaaatg caaccctaat 1600ttacattact ggactactca ggatgaaggt gctgcaatcg 1640 gactggcctg gataccatatttcgggccag cagccgaggg 1680 aatttacata gaggggctaa tgcacaatca agatggttta1720 atctgtgggt tgagacagct ggccaacgag acgactcaag 1760 ctcttcaactgttcctgaga gccacaactg agctacgcac 1800 cttttcaatc ctcaaccgta aggcaattgatttcttgctg 1840 cagcgatggg gcggcacatg ccacattctg ggaccggact 1880gctgtatcga accacatgat tggaccaaga acataacaga 1920 caaaattgat cagattattcatgattttgt tgataaaacc 1960 cttccggacc agggggacaa tgacaattgg tggacaggat2000 ggagacaatg gataccggca ggtattggag ttacaggcgt 2040 tgtaattgcagttatcgctt tattctgtat atgcaaattt 2080 gtcttttagt ttttcttcag attgcttcatggaaaagctc 2120 agcctcaaat caatgaaacc aggatttaat tatatggatt 2160acttgaatct aagattactt gacaaatgat aatataatac 2200 actggagctt taaacatagccaatgtgatt ctaactcctt 2240 taaactcaca gttaatcata aacaaggttt gagtcgacct2280 gcagccaagc ttatcgat 2298 2 2428 DNA Ebola Zaire 2 atcgataagcttggctgcag gtcgactcta gaggatccga 40 gtatggattc tcgtcctcag aaaatctggatggcgccgag 80 tctcactgaa tctgacatgg attaccacaa gatcttgaca 120 gcaggtctgtccgttcaaca ggggattgtt cggcaaagag 160 tcatcccagt gtatcaagta aacaatcttgaagaaatttg 200 ccaacttatc atacaggcct ttgaagcagg tgttgatttt 240caagagagtg cggacagttt ccttctgatg ctttgtcttc 280 atcatgcgta ccagggagattacaaacttt tcttggaaag 320 tggcgcagtc aagtatttgg aagggcacgg gttccgtttt360 gaagtcaaga agcgtgatgg agtgaagcga cttgaggaat 400 tgctgccagcagtatctagt ggaaaaaaca ttaagagaac 440 acttgctgcc atgccggaag aggagacaactgaagctaat 480 gccggtcagt ttctctcctt tgcaagtcta ttccttccga 520aattggtagt aggagaaaag gcttgccttg agaaggttca 560 aaggcaaatt caagtacatgcagagcaagg actgatacaa 600 tatccaacag cttggcaatc agtaggacac atgatggtga640 ttttccgttt gatgcgaaca aattttctga tcaaatttct 680 cctaatacaccaagggatgc acatggttgc cgggcatgat 720 gccaacgatg ctgtgatttc aaattcagtggctcaagctc 760 gtttttcagg cttattgatt gtcaaaacag tacttgatca 800tatcctacaa aagacagaac gaggagttcg tctccatcct 840 cttgcaagga ccgccaaggtaaaaaatgag gtgaactcct 880 ttaaggctgc actcagctcc ctggccaagc atggagagta920 tgctcctttc gcccgacttt tgaacctttc tggagtaaat 960 aatcttgagcatggtctttt ccctcaacta tcggcaattg 1000 cactcggagt cgccacagca cacgggagtaccctcgcagg 1040 agtaaatgtt ggagaacagt atcaacaact cagagaggct 1080gccactgagg ctgagaagca actccaacaa tatgcagagt 1120 ctcgcgaact tgaccatcttggacttgatg atcaggaaaa 1160 gaaaattctt atgaacttcc atcagaaaaa gaacgaaatc1200 agcttccagc aaacaaacgc tatggtaact ctaagaaaag 1240 agcgcctggccaagctgaca gaagctatca ctgctgcgtc 1280 actgcccaaa acaagtggac attacgatgatgatgacgac 1320 attccctttc caggacccat caatgatgac gacaatccta 1360gccatcaaga tgatgatccg actgactcac aggatacgac 1400 cattcccgat gtggtggttgatcccgatga tggaagctac 1440 ggcgaatacc agagttactc ggaaaacggc atgaatgcac1480 cagatgactt ggtcctattc gatctagacg aggacgacga 1520 ggacactaagccagtgccta atagatcgac caagggtgga 1560 caacagaaga acagtcaaaa gggccagcatatagagggca 1600 gacagacaca atccaggcca attcaaaatg tcccaggccc 1640tcacagaaca atccaccacg ccagtgcgcc actcacggac 1680 aatgacagaa gaaatgaaccctccggctca accagccctc 1720 gcatgctgac accaattaac gaagaggcag acccactgga1760 cgatgccgac gacgagacgt ctagccttcc gcccttggag 1800 tcagatgatgaagagcagga cagggacgga acttccaacc 1840 gcacacccac tgtcgcccca ccggctcccgtatacagaga 1880 tcactctgaa aagaaagaac tcccgcaaga cgagcaacaa 1920gatcaggacc acactcaaga ggccaggaac caggacagtg 1960 acaacaccca gtcagaacactcttttgagg agatgtatcg 2000 ccacattcta agatcacagg ggccatttga tgctgttttg2040 tattatcata tgatgaagga tgagcctgta gttttcagta 2080 ccagtgatggcaaagagtac acgtatccag actcccttga 2120 agaggaatat ccaccatggc tcactgaaaaagaggctatg 2160 aatgaagaga atagatttgt tacattggat ggtcaacaat 2200tttattggcc ggtgatgaat cacaagaata aattcatggc 2240 aatcctgcaa catcatcagtgaatgagcat ggaacaatgg 2280 gatgattcaa ccgacaaata gctaacatta agtagtccag2320 gaacgaaaac aggaagaatt tttgatgtct aaggtgtgaa 2360 ttattatcacaataaaagtg attcttattt ttgaatttgg 2400 gcgagctcga attcccgagc ttatcgat2428 3 847 DNA Ebola Zaire 3 atcgatctcc agacaccaag caagacctga gaaaaaacca40 tggctaaagc tacgggacga tacaatctaa tatcgcccaa 80 aaaggacctg gagaaaggggttgtcttaag cgacctctgt 120 aacttcttag ttagccaaac tattcagggg tggaaggttt160 attgggctgg tattgagttt gatgtgactc acaaaggaat 200 ggccctattgcatagactga aaactaatga ctttgcccct 240 gcatggtcaa tgacaaggaa tctctttcctcatttatttc 280 aaaatccgaa ttccacaatt gaatcaccgc tgtgggcatt 320gagagtcatc cttgcagcag ggatacagga ccagctgatt 360 gaccagtctt tgattgaacccttagcagga gcccttggtc 400 tgatctctga ttggctgcta acaaccaaca ctaaccattt440 caacatgcga acacaacgtg tcaaggaaca attgagccta 480 aaaatgctgtcgttgattcg atccaatatt ctcaagttta 520 ttaacaaatt ggatgctcta catgtcgtgaactacaacgg 560 attgttgagc agtattgaaa ttggaactca aaatcataca 600atcatcataa ctcgaactaa catgggtttt ctggtggagc 640 tccaagaacc cgacaaatcggcaatgaacc gcatgaagcc 680 tgggccggcg aaattttccc tccttcatga gtccacactg720 aaagcattta cacaaggatc ctcgacacga atgcaaagtt 760 tgattcttgaatttaatagc tctcttgcta tctaactaag 800 gtagaatact tcatattgag ctaactcatatatgctgact 840 catcgat 847 4 973 DNA Ebola Zaire 4 atcgatcaga tctgcgaaccggtagagttt agttgcaacc 40 taacacacat aaagcattgg tcaaaaagtc aatagaaatt 80taaacagtga gtggagacaa cttttaaatg gaagcttcat 120 atgagagagg acgcccacgagctgccagac agcattcaag 160 ggatggacac gaccaccatg ttcgagcacg atcatcatcc200 agagagaatt atcgaggtga gtaccgtcaa tcaaggagcg 240 cctcacaagtgcgcgttcct actgtatttc ataagaagag 280 agttgaacca ttaacagttc ctccagcacctaaagacata 320 tgtccgacct tgaaaaaagg atttttgtgt gacagtagtt 360tttgcaaaaa agatcaccag ttggagagtt taactgatag 400 ggaattactc ctactaatcgcccgtaagac ttgtggatca 440 gtagaacaac aattaaatat aactgcaccc aaggactcgc480 gcttagcaaa tccaacggct gatgatttcc agcaagagga 520 aggtccaaaaattaccttgt tgacactgat caagacggca 560 gaacactggg cgagacaaga catcagaaccatagaggatt 600 caaaattaag agcattgttg actctatgtg ctgtgatgac 640gaggaaattc tcaaaatccc agctgagtct tttatgtgag 680 acacacctaa ggcgcgaggggcttgggcaa gatcaggcag 720 aacccgttct cgaagtatat caacgattac acagtgataa760 aggaggcagt tttgaagctg cactatggca acaatgggac 800 ctacaatccctaattatgtt tatcactgca ttcttgaata 840 ttgctctcca gttaccgtgt gaaagttctgctgtcgttgt 880 ttcagggtta agaacattgg ttcctcaatc agataatgag 920gaagcttcaa ccaacccggg gacatgctca tggtctgatg 960 agggtacatc gat 973 51148 DNA Ebola Zaire 5 atcgatagaa aagctggtct aacaagatga caactagaac 40aaagggcagg ggccatactg cggccacgac tcaaaacgac 80 agaatgccag gccctgagctttcgggctgg atctctgagc 120 agctaatgac cggaagaatt cctgtaagcg acatcttctg160 tgatattgag aacaatccag gattatgcta cgcatcccaa 200 atgcaacaaacgaagccaaa cccgaagacg cgcaacagtc 240 aaacccaaac ggacccaatt tgcaatcatagttttgagga 280 ggtagtacaa acattggctt cattggctac tgttgtgcaa 320caacaaacca tcgcatcaga atcattagaa caacgcatta 360 cgagtcttga gaatggtctaaagccagttt atgatatggc 400 aaaaacaatc tcctcattga acagggtttg tgctgagatg440 gttgcaaaat atgatcttct ggtgatgaca accggtcggg 480 caacagcaaccgctgcggca actgaggctt attgggccga 520 acatggtcaa ccaccacctg gaccatcactttatgaagaa 560 agtgcgattc ggggtaagat tgaatctaga gatgagaccg 600tccctcaaag tgttagggag gcattcaaca atctaaacag 640 taccacttca ctaactgaggaaaattttgg gaaacctgac 680 atttcggcaa aggatttgag aaacattatg tatgatcact720 tgcctggttt tggaactgct ttccaccaat tagtacaagt 760 gatttgtaaattgggaaaag atagcaactc attggacatc 800 attcatgctg agttccaggc cagcctggctgaaggagact 840 ctcctcaatg tgccctaatt caaattacaa aaagagttcc 880aatcttccaa gatgctgctc cacctgtcat ccacatccgc 920 tctcgaggtg acattccccgagcttgccag aaaagcttgc 960 gtccagtccc accatcgccc aagattgatc gaggttgggt1000 atgtgttttt cagcttcaag atggtaaaac acttggactc 1040 aaaatttgagccaatctccc ttccctccga aagaggcgaa 1080 taatagcaga ggcttcaact gctgaactatagggtacgtt 1120 acattaatga tacacttgtg agatcgat 1148 6 1123 DNA EbolaZaire 6 atcgatccta cctcggctga gagagtgttt tttcattaac 40 cttcatcttgtaaacgttga gcaaaattgt taaaaatatg 80 aggcgggtta tattgcctac tgctcctcctgaatatatgg 120 aggccatata ccctgtcagg tcaaattcaa caattgctag 160aggtggcaac agcaatacag gcttcctgac accggagtca 200 gtcaatgggg acactccatcgaatccactc aggccaattg 240 ccgatgacac catcgaccat gccagccaca caccaggcag280 tgtgtcatca gcattcatcc ttgaagctat ggtgaatgtc 320 atatcgggccccaaagtgct aatgaagcaa attccaattt 360 ggcttcctct aggtgtcgct gatcaaaagacctacagctt 400 tgactcaact acggccgcca tcatgcttgc ttcatacact 440atcacccatt tcggcaaggc aaccaatcca cttgtcagag 480 tcaatcggct gggtcctggaatcccggatc atcccctcag 520 gctcctgcga attggaaacc aggctttcct ccaggagttc560 gttcttccgc cagtccaact accccagtat ttcacctttg 600 atttgacagcactcaaactg atcacccaac cactgcctgc 640 tgcaacatgg accgatgaca ctccaacaggatcaaatgga 680 gcgttgcgtc caggaatttc atttcatcca aaacttcgcc 720ccattctttt acccaacaaa agtgggaaga aggggaacag 760 tgccgatcta acatctccggagaaaatcca agcaataatg 800 acttcactcc aggactttaa gatcgttcca attgatccaa840 ccaaaaatat catgggaatc gaagtgccag aaactctggt 880 ccacaagctgaccggtaaga aggtgacttc taaaaatgga 920 caaccaatca tccctgttct tttgccaaagtacattgggt 960 tggacccggt ggctccagga gacctcacca tggtaatcac 1000acaggattgt gacacgtgtc attctcctgc aagtcttcca 1040 gctgtgattg agaagtaattgcaataattg actcagatcc 1080 agttttatag aatcttctca gggatagtgc ataacatatc1120 gat 1123 7 1165 DNA Ebola Zaire 7 atcgatcaga tctgcgaacc ggtagagtttagttgcaacc 40 taacacacat aaagcattgg tcaaaaagtc aatagaaatt 80 taaacagtgagtggagacaa cttttaaatg gaagcttcat 120 atgagagagg acgcccacga gctgccagacagcattcaag 160 ggatggacac gaccaccatg ttcgagcacg atcatcatcc 200agagagaatt atcgaggtga gtaccgtcaa tcaaggagcg 240 cctcacaagt gcgcgttcctactgtatttc ataagaagag 280 agttgaacca ttaacagttc ctccagcacc taaagacata320 tgtccgacct tgaaaaaagg atttttgtgt gacagtagtt 360 tttgcaaaaaagatcaccag ttggagagtt taactgatag 400 ggaattactc ctactaatcg cccgtaagacttgtggatca 440 gtagaacaac aattaaatat aactgcaccc aaggactcgc 480gcttagcaaa tccaacggct gatgatttcc agcaagagga 520 aggtccaaaa attaccttgttgacactgat caagacggca 560 gaacactggg cgagacaaga catcagaacc atagaggatt600 caaaattaag agcattgttg actctatgtg ctgtgatgac 640 gaggaaattctcaaaatccc agctgagtct tttatgtgag 680 acacacctaa ggcgcgaggg gcttgggcaagatcaggcag 720 aacccgttct cgaagtatat caacgattac acagtgataa 760aggaggcagt tttgaagctg cactatggca acaatgggac 800 cgacaatccc taatcatgtttatcactgca ttcttgaata 840 ttgctctcca gttaccgtgt gaaagttctg ctgtcgttgt880 ttcagggtta agaacattgg ttcctcaatc agataatgag 920 gaagcttcaaccaacccggg gacatgctca tggtctgatg 960 agggtacccc ttaataaggc tgactaaaacactatataac 1000 cttctacttg atcacaatac tccgtatacc tatcatcata 1040tatttaatca agacgatatc ctttaaaact tattcagtac 1080 tataatcact ctcgtttcaaattaataaga tgtgcatgat 1120 tgccctaata tatgaagagg tatgatacaa ccctaacaga1160 tcgat 1165 8 30 DNA artificial sequence /note= “forward primer forVP24” 8 gggatcgatc tccagacacc aagcaagacc 30 9 33 DNA artificial sequence/note= “reverse primer for VP24” 9 gggatcgatg agtcagcata tatgagttag ctc33 10 30 DNA artificial sequence /note= “forward primer for VP30” 10cccatcgatc agatctgcga accggtagag 30 11 31 DNA artificial sequence /note=“reverse primer for VP30” 11 cccatcgatg taccctcatc agaccatgag c 31 12 33DNA artificial sequence /note= “forward primer for VP35” 12 gggatcgatagaaaagctgg tctaacaaga tga 33 13 36 DNA artificial sequence /note=“reverse primer for VP35” 13 cccatcgatc tcacaagtgt atcattaatg taacgt 3614 30 DNA artificial sequence /note= “forward primer for VP40” 14cccatcgatc ctacctcggc tgagagagtg 30 15 33 DNA artificial sequence /note=“reverse primer for VP40” 15 cccatcgata tgttatgcac tatccctgag aag 33 1630 DNA artificial sequence /note= “reverse primer for VP30#2” 16cccatcgatc tgttagggtt gtatcatacc 30 17 676 PRT Ebola Zaire 17 Met GlyVal Thr Gly Ile Leu Gln Leu Pro 1 5 10 Arg Asp Arg Phe Lys Arg Thr SerPhe Phe 15 20 Leu Trp Val Ile Ile Leu Phe Gln Arg Thr 25 30 Phe Ser IlePro Leu Gly Val Ile His Asn 35 40 Ser Thr Leu Gln Val Ser Asp Val AspLys 45 50 Leu Val Cys Arg Asp Lys Leu Ser Ser Thr 55 60 Asn Gln Leu ArgSer Val Gly Leu Asn Leu 65 70 Glu Gly Asn Gly Val Ala Thr Asp Val Pro 7580 Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser 85 90 Gly Val Pro Pro Lys ValVal Asn Tyr Glu 95 100 Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn 105 110Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu 115 120 Cys Leu Pro Ala Ala ProAsp Gly Ile Arg 125 130 Gly Phe Pro Arg Cys Arg Tyr Val His Lys 135 140Val Ser Gly Thr Gly Pro Cys Ala Gly Asp 145 150 Phe Ala Phe His Lys GluGly Ala Phe Phe 155 160 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile 165 170Tyr Arg Gly Thr Thr Phe Ala Glu Gly Val 175 180 Val Ala Phe Leu Ile LeuPro Gln Ala Lys 185 190 Lys Asp Phe Phe Ser Ser His Pro Leu Arg 195 200Glu Pro Val Asn Ala Thr Glu Asp Pro Ser 205 210 Ser Gly Tyr Tyr Ser ThrThr Ile Arg Tyr 215 220 Gln Ala Thr Gly Phe Gly Thr Asn Glu Thr 225 230Glu Tyr Leu Phe Glu Val Asp Asn Leu Thr 235 240 Tyr Val Gln Leu Glu SerArg Phe Thr Pro 245 250 Gln Phe Leu Leu Gln Leu Asn Glu Thr Ile 255 260Tyr Thr Ser Gly Lys Arg Ser Asn Thr Thr 265 270 Gly Lys Leu Ile Trp LysVal Asn Pro Glu 275 280 Ile Asp Thr Thr Ile Gly Glu Trp Ala Phe 285 290Trp Glu Thr Lys Lys Asn Leu Thr Arg Lys 295 300 Ile Arg Ser Glu Glu LeuSer Phe Thr Val 305 310 Val Ser Asn Gly Ala Lys Asn Ile Ser Gly 315 320Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro 325 330 Gly Thr Asn Thr Thr ThrGlu Asp His Lys 335 340 Ile Met Ala Ser Glu Asn Ser Ser Ala Met 345 350Val Gln Val His Ser Gln Gly Arg Glu Ala 355 360 Ala Val Ser His Leu ThrThr Leu Ala Thr 365 370 Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr 375 380Lys Pro Gly Pro Asp Asn Ser Thr His Asn 385 390 Thr Pro Val Tyr Lys LeuAsp Ile Ser Glu 395 400 Ala Thr Gln Val Glu Gln His His Arg Arg 405 410Thr Asp Asn Asp Ser Thr Ala Ser Asp Thr 415 420 Pro Ser Ala Thr Thr AlaAla Gly Pro Pro 425 430 Lys Ala Glu Asn Thr Asn Thr Ser Lys Ser 435 440Thr Asp Phe Leu Asp Pro Ala Thr Thr Thr 445 450 Ser Pro Gln Asn His SerGlu Thr Ala Gly 455 460 Asn Asn Asn Thr His His Gln Asp Thr Gly 465 470Glu Glu Ser Ala Ser Ser Gly Lys Leu Gly 475 480 Leu Ile Thr Asn Thr IleAla Gly Val Ala 485 490 Gly Leu Ile Thr Gly Gly Arg Arg Thr Arg 495 500Arg Glu Ala Ile Val Asn Ala Gln Pro Lys 505 510 Cys Asn Pro Asn Leu HisTyr Trp Thr Thr 515 520 Gln Asp Glu Gly Ala Ala Ile Gly Leu Ala 525 530Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu 535 540 Gly Ile Tyr Ile Glu GlyLeu Met His Asn 545 550 Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln 555 560Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln 565 570 Leu Phe Leu Arg Ala ThrThr Glu Leu Arg 575 580 Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile 585 590Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr 595 600 Cys His Ile Leu Gly ProAsp Cys Cys Ile 605 610 Glu Pro His Asp Trp Thr Lys Asn Ile Thr 615 620Asp Lys Ile Asp Gln Ile Ile His Asp Phe 625 630 Val Asp Lys Thr Leu ProAsp Gln Gly Asp 635 640 Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln 645 650Trp Ile Pro Ala Gly Ile Gly Val Thr Gly 655 660 Val Val Ile Ala Val IleAla Leu Phe Cys 665 670 Ile Cys Lys Phe Val Phe 675 18 739 PRT EbolaZaire 18 Met Asp Ser Arg Pro Gln Lys Ile Trp Met 1 5 10 Ala Pro Ser LeuThr Glu Ser Asp Met Asp 15 20 Tyr His Lys Ile Leu Thr Ala Gly Leu Ser 2530 Val Gln Gln Gly Ile Val Arg Gln Arg Val 35 40 Ile Pro Val Tyr Gln ValAsn Asn Leu Glu 45 50 Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe 55 60 GluAla Gly Val Asp Phe Gln Glu Ser Ala 65 70 Asp Ser Phe Leu Leu Met LeuCys Leu His 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe 85 90 Leu GluSer Gly Ala Val Lys Tyr Leu Glu 95 100 Gly His Gly Phe Arg Phe Glu ValLys Lys 105 110 Arg Asp Gly Val Lys Arg Leu Glu Glu Leu 115 120 Leu ProAla Val Ser Ser Gly Lys Asn Ile 125 130 Lys Arg Thr Leu Ala Ala Met ProGlu Glu 135 140 Glu Thr Thr Glu Ala Asn Ala Gly Gln Phe 145 150 Leu SerPhe Ala Ser Leu Phe Leu Pro Lys 155 160 Leu Val Val Gly Glu Lys Ala CysLeu Glu 165 170 Lys Val Gln Arg Gln Ile Gln Val His Ala 175 180 Glu GlnGly Leu Ile Gln Tyr Pro Thr Ala 185 190 Trp Gln Ser Val Gly His Met MetVal Ile 195 200 Phe Arg Leu Met Arg Thr Asn Phe Leu Ile 205 210 Lys PheLeu Leu Ile His Gln Gly Met His 215 220 Met Val Ala Gly His Asp Ala AsnAsp Ala 225 230 Val Ile Ser Asn Ser Val Ala Gln Ala Arg 235 240 Phe SerGly Leu Leu Ile Val Lys Thr Val 245 250 Leu Asp His Ile Leu Gln Lys ThrGlu Arg 255 260 Gly Val Arg Leu His Pro Leu Ala Arg Thr 265 270 Ala LysVal Lys Asn Glu Val Asn Ser Phe 275 280 Lys Ala Ala Leu Ser Ser Leu AlaLys His 285 290 Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu 295 300 Asn LeuSer Gly Val Asn Asn Leu Glu His 305 310 Gly Leu Phe Pro Gln Leu Ser AlaIle Ala 315 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr 325 330 Leu AlaGly Val Asn Val Gly Glu Gln Tyr 335 340 Gln Gln Leu Arg Glu Ala Ala ThrGlu Ala 345 350 Glu Lys Gln Leu Gln Gln Tyr Ala Glu Ser 355 360 Arg GluLeu Asp His Leu Gly Leu Asp Asp 365 370 Gln Glu Lys Lys Ile Leu Met AsnPhe His 375 380 Gln Lys Lys Asn Glu Ile Ser Phe Gln Gln 385 390 Thr AsnAla Met Val Thr Leu Arg Lys Glu 395 400 Arg Leu Ala Lys Leu Thr Glu AlaIle Thr 405 410 Ala Ala Ser Leu Pro Lys Thr Ser Gly His 415 420 Tyr AspAsp Asp Asp Asp Ile Pro Phe Pro 425 430 Gly Pro Ile Asn Asp Asp Asp AsnPro Gly 435 440 His Gln Asp Asp Asp Pro Thr Asp Ser Gln 445 450 Asp ThrThr Ile Pro Asp Val Val Val Asp 455 460 Pro Asp Asp Gly Ser Tyr Gly GluTyr Gln 465 470 Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro 475 480 Asp AspLeu Val Leu Phe Asp Leu Asp Glu 485 490 Asp Asp Glu Asp Thr Lys Pro ValPro Asn 495 500 Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn 505 510 Ser GlnLys Gly Gln His Ile Glu Gly Arg 515 520 Gln Thr Gln Ser Arg Pro Ile GlnAsn Val 525 530 Pro Gly Pro His Arg Thr Ile His His Ala 535 540 Ser AlaPro Leu Thr Asp Asn Asp Arg Arg 545 550 Asn Glu Pro Ser Gly Ser Thr SerPro Arg 555 560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp 565 570 Pro LeuAsp Asp Ala Asp Asp Glu Thr Ser 575 580 Ser Leu Pro Pro Leu Glu Ser AspAsp Glu 585 590 Glu Gln Asp Arg Asp Gly Thr Ser Asn Arg 595 600 Thr ProThr Val Ala Pro Pro Ala Pro Val 605 610 Tyr Arg Asp His Ser Glu Lys LysGlu Leu 615 620 Pro Gln Asp Glu Gln Gln Asp Gln Asp His 625 630 Thr GlnGlu Ala Arg Asn Gln Asp Ser Asp 635 640 Asn Thr Gln Ser Glu His Ser PheGlu Glu 645 650 Met Tyr Arg His Ile Leu Arg Ser Gln Gly 655 660 Pro PheAsp Ala Val Leu Tyr Tyr His Met 665 670 Met Lys Asp Glu Pro Val Val PheSer Thr 675 680 Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp 685 690 Ser LeuGlu Glu Glu Tyr Pro Pro Trp Leu 695 700 Thr Glu Lys Glu Ala Met Asn GluGlu Asn 705 710 Arg Phe Val Thr Leu Asp Gly Gln Gln Phe 715 720 Tyr TrpPro Val Met Asn His Lys Asn Lys 725 730 Phe Met Ala Ile Leu Gln His HisGln 735 19 251 PRT Ebola Zaire 19 Met Ala Lys Ala Thr Gly Arg Tyr AsnLeu 1 5 10 Ile Ser Pro Lys Lys Asp Leu Glu Lys Gly 15 20 Val Val Leu SerAsp Leu Cys Asn Phe Leu 25 30 Val Ser Gln Thr Ile Gln Gly Trp Lys Val 3540 Tyr Trp Ala Gly Ile Glu Phe Asp Val Thr 45 50 His Lys Gly Met Ala LeuLeu His Arg Leu 55 60 Lys Thr Asn Asp Phe Ala Pro Ala Trp Ser 65 70 MetThr Arg Asn Leu Phe Pro His Leu Phe 75 80 Gln Asn Pro Asn Ser Thr IleGlu Ser Pro 85 90 Leu Trp Ala Leu Arg Val Ile Leu Ala Ala 95 100 Gly IleGln Asp Gln Leu Ile Asp Gln Ser 105 110 Leu Ile Glu Pro Leu Ala Gly AlaLeu Gly 115 120 Leu Ile Ser Asp Trp Leu Leu Thr Thr Asn 125 130 Thr AsnHis Phe Asn Met Arg Thr Gln Arg 135 140 Val Lys Glu Gln Leu Ser Leu LysMet Leu 145 150 Ser Leu Ile Arg Ser Asn Ile Leu Lys Phe 155 160 Ile AsnLys Leu Asp Ala Leu His Val Val 165 170 Asn Tyr Asn Gly Leu Leu Ser SerIle Glu 175 180 Ile Gly Thr Gln Asn His Thr Ile Ile Ile 185 190 Thr ArgThr Asn Met Gly Phe Leu Val Glu 195 200 Leu Gln Glu Pro Asp Lys Ser AlaMet Asn 205 210 Arg Met Lys Pro Gly Pro Ala Lys Phe Ser 215 220 Leu LeuHis Glu Ser Thr Leu Lys Ala Phe 225 230 Thr Gln Gly Ser Ser Thr Arg MetGln Ser 235 240 Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala 245 250 Ile 20324 PRT Ebola Zaire 20 Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro 1 5 10Arg Ala Ala Arg Gln His Ser Arg Asp Gly 15 20 His Asp His His Val ArgAla Arg Ser Ser 25 30 Ser Arg Glu Asn Tyr Arg Gly Glu Tyr Arg 35 40 GlnSer Arg Ser Ala Ser Gln Val Arg Val 45 50 Pro Thr Val Phe His Lys LysArg Val Glu 55 60 Pro Leu Thr Val Pro Pro Ala Pro Lys Asp 65 70 Ile CysPro Thr Leu Lys Lys Gly Phe Leu 75 80 Cys Asp Ser Ser Phe Cys Lys LysAsp His 85 90 Gln Leu Glu Ser Leu Thr Asp Arg Glu Leu 95 100 Leu Leu LeuIle Ala Arg Lys Thr Cys Gly 105 110 Ser Val Glu Gln Gln Leu Asn Ile ThrAla 115 120 Pro Lys Asp Ser Arg Leu Ala Asn Pro Thr 125 130 Ala Asp AspPhe Gln Gln Glu Glu Gly Pro 135 140 Lys Ile Thr Leu Leu Thr Leu Ile LysThr 145 150 Ala Glu His Trp Ala Arg Gln Asp Ile Arg 155 160 Thr Ile GluAsp Ser Lys Leu Arg Ala Leu 165 170 Leu Thr Leu Cys Ala Val Met Thr ArgLys 175 180 Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys 185 190 Glu Thr HisLeu Arg Arg Glu Gly Leu Gly 195 200 Gln Asp Gln Ala Glu Pro Val Leu GluVal 205 210 Tyr Gln Arg Leu His Ser Asp Lys Gly Gly 215 220 Ser Phe GluAla Ala Leu Trp Gln Gln Trp 225 230 Asp Leu Gln Ser Leu Ile Met Phe IleThr 235 240 Ala Phe Leu Asn Ile Ala Leu Gln Leu Pro 245 250 Cys Glu SerSer Ala Val Val Val Ser Gly 255 260 Leu Arg Thr Leu Val Pro Gln Ser AspAsn 265 270 Glu Glu Ala Ser Thr Asn Pro Gly Thr Cys 275 280 Ser Trp SerAsp Glu Gly Thr Ser Ile Gln 285 290 Gln Gln Leu Ala Ser Cys Leu His ArgThr 295 300 Arg Gly Asp Trp His Ala Ala Leu Lys Phe 305 310 Leu Phe TyrPhe Ser Phe Leu Phe Arg Ile 315 320 Gly Phe Cys Phe 21 340 PRT EbolaZaire 21 Met Thr Thr Arg Thr Lys Gly Arg Gly His 1 5 10 Thr Ala Ala ThrThr Gln Asn Asp Arg Met 15 20 Pro Gly Pro Glu Leu Ser Gly Trp Ile Ser 2530 Glu Gln Leu Met Thr Gly Arg Ile Pro Val 35 40 Ser Asp Ile Phe Cys AspIle Glu Asn Asn 45 50 Pro Gly Leu Cys Tyr Ala Ser Gln Met Gln 55 60 GlnThr Lys Pro Asn Pro Lys Thr Arg Asn 65 70 Ser Gln Thr Gln Thr Asp ProIle Cys Asn 75 80 His Ser Phe Glu Glu Val Val Gln Thr Leu 85 90 Ala SerLeu Ala Thr Val Val Gln Gln Gln 95 100 Thr Ile Ala Ser Glu Ser Leu GluGln Arg 105 110 Ile Thr Ser Leu Glu Asn Gly Leu Lys Pro 115 120 Val TyrAsp Met Ala Lys Thr Ile Ser Ser 125 130 Leu Asn Arg Val Cys Ala Glu MetVal Ala 135 140 Lys Tyr Asp Leu Leu Val Met Thr Thr Gly 145 150 Arg AlaThr Ala Thr Ala Ala Ala Thr Glu 155 160 Ala Tyr Trp Ala Glu His Gly GlnPro Pro 165 170 Pro Gly Pro Ser Leu Tyr Glu Glu Ser Ala 175 180 Ile ArgGly Lys Ile Glu Ser Arg Asp Glu 185 190 Thr Val Pro Gln Ser Val Arg GluAla Phe 195 200 Asn Asn Leu Asn Ser Thr Thr Ser Leu Thr 205 210 Glu GluAsn Phe Gly Lys Pro Asp Ile Ser 215 220 Ala Lys Asp Leu Arg Asn Ile MetTyr Asp 225 230 His Leu Pro Gly Phe Gly Thr Ala Phe His 235 240 Gln LeuVal Gln Val Ile Cys Lys Leu Gly 245 250 Lys Asp Ser Asn Ser Leu Asp IleIle His 255 260 Ala Glu Phe Gln Ala Ser Leu Ala Glu Gly 265 270 Asp SerPro Gln Cys Ala Leu Ile Gln Ile 275 280 Thr Lys Arg Val Pro Ile Phe GlnAsp Ala 285 290 Ala Pro Pro Val Ile His Ile Arg Ser Arg 295 300 Gly AspIle Pro Arg Ala Cys Gln Lys Ser 305 310 Leu Arg Pro Val Pro Pro Ser ProLys Ile 315 320 Asp Arg Gly Trp Val Cys Val Phe Gln Leu 325 330 Gln AspGly Lys Thr Leu Gly Leu Lys Ile 335 340 22 326 PRT Ebola Zaire 22 MetArg Arg Val Ile Leu Pro Thr Ala Pro 1 5 10 Pro Glu Tyr Met Glu Ala IleTyr Pro Val 15 20 Arg Ser Asn Ser Thr Ile Ala Arg Gly Gly 25 30 Asn SerAsn Thr Gly Phe Leu Thr Pro Glu 35 40 Ser Val Asn Gly Asp Thr Pro SerAsn Pro 45 50 Leu Arg Pro Ile Ala Asp Asp Thr Ile Asp 55 60 His Ala SerHis Thr Pro Gly Ser Val Ser 65 70 Ser Ala Phe Ile Leu Glu Ala Met ValAsn 75 80 Val Ile Ser Gly Pro Lys Val Leu Met Lys 85 90 Gln Ile Pro IleTrp Leu Pro Leu Gly Val 95 100 Ala Asp Gln Lys Thr Tyr Ser Phe Asp Ser105 110 Thr Thr Ala Ala Ile Met Leu Ala Ser Tyr 115 120 Thr Ile Thr HisPhe Gly Lys Ala Thr Asn 125 130 Pro Leu Val Arg Val Asn Arg Leu Gly Pro135 140 Gly Ile Pro Asp His Pro Leu Arg Leu Leu 145 150 Arg Ile Gly AsnGln Ala Phe Leu Gln Glu 155 160 Phe Val Leu Pro Pro Val Gln Leu Pro Gln165 170 Tyr Phe Thr Phe Asp Leu Thr Ala Leu Lys 175 180 Leu Ile Thr GlnPro Leu Pro Ala Ala Thr 185 190 Trp Thr Asp Asp Thr Pro Thr Gly Ser Asn195 200 Gly Ala Leu Arg Pro Gly Ile Ser Phe His 205 210 Pro Lys Leu ArgPro Ile Leu Leu Pro Asn 215 220 Lys Ser Gly Lys Lys Gly Asn Ser Ala Asp225 230 Leu Thr Ser Pro Glu Lys Ile Gln Ala Ile 235 240 Met Thr Ser LeuGln Asp Phe Lys Ile Val 245 250 Pro Ile Asp Pro Thr Lys Asn Ile Met Gly255 260 Ile Glu Val Pro Glu Thr Leu Val His Lys 265 270 Leu Thr Gly LysLys Val Thr Ser Lys Asn 275 280 Gly Gln Pro Ile Ile Pro Val Leu Leu Pro285 290 Lys Tyr Ile Gly Leu Asp Pro Val Ala Pro 295 300 Gly Asp Leu ThrMet Val Ile Thr Gln Asp 305 310 Cys Asp Thr Cys His Ser Pro Ala Ser Leu315 320 Pro Ala Val Ile Glu Lys 325 23 288 PRT Ebola Zaire 23 Met GluAla Ser Tyr Glu Arg Gly Arg Pro 1 5 10 Arg Ala Ala Arg Gln His Ser ArgAsp Gly 15 20 His Asp His His Val Arg Ala Arg Ser Ser 25 30 Ser Arg GluAsn Tyr Arg Gly Glu Tyr Arg 35 40 Gln Ser Arg Ser Ala Ser Gln Val ArgVal 45 50 Pro Thr Val Phe His Lys Lys Arg Val Glu 55 60 Pro Leu Thr ValPro Pro Ala Pro Lys Asp 65 70 Ile Cys Pro Thr Leu Lys Lys Gly Phe Leu 7580 Cys Asp Ser Ser Phe Cys Lys Lys Asp His 85 90 Gln Leu Glu Ser Leu ThrAsp Arg Glu Leu 95 100 Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly 105 110Ser Val Glu Gln Gln Leu Asn Ile Thr Ala 115 120 Pro Lys Asp Ser Arg LeuAla Asn Pro Thr 125 130 Ala Asp Asp Phe Gln Gln Glu Glu Gly Pro 135 140Lys Ile Thr Leu Leu Thr Leu Ile Lys Thr 145 150 Ala Glu His Trp Ala ArgGln Asp Ile Arg 155 160 Thr Ile Glu Asp Ser Lys Leu Arg Ala Leu 165 170Leu Thr Leu Cys Ala Val Met Thr Arg Lys 175 180 Phe Ser Lys Ser Gln LeuSer Leu Leu Cys 185 190 Glu Thr His Leu Arg Arg Glu Gly Leu Gly 195 200Gln Asp Gln Ala Glu Pro Val Leu Glu Val 205 210 Tyr Gln Arg Leu His SerAsp Lys Gly Gly 215 220 Ser Phe Glu Ala Ala Leu Trp Gln Gln Trp 225 230Asp Arg Gln Ser Leu Ile Met Phe Ile Thr 235 240 Ala Phe Leu Asn Ile AlaLeu Gln Leu Pro 245 250 Cys Glu Ser Ser Ala Val Val Val Ser Gly 255 260Leu Arg Thr Leu Val Pro Gln Ser Asp Asn 265 270 Glu Glu Ala Ser Thr AsnPro Gly Thr Cys 275 280 Ser Trp Ser Asp Glu Gly Thr Pro 285 24 11 PRTEbola Zaire 24 Val Tyr Gln Val Asn Asn Leu Glu Glu Ile 1 5 10 Cys 25 23PRT Ebola Zaire 25 Leu Lys Phe Ile Asn Lys Leu Asp Ala Leu 1 5 10 LeuVal Val Asn Tyr Asn Gly Leu Leu Ser 15 20 Ser Ile Phe

What is claimed is:
 1. A DNA fragment which encodes a GP Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO:1, or a polynucleotide fragment comprising at least 15 nucleotides.
 2. A DNA fragment which encodes a NP Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO:2, or a polynucleotide fragment comprising at least 15 nucleotides.
 3. A DNA fragment which encodes a VP24 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO:3, or a polynucleotide fragment comprising at least 15 nucleotides.
 4. A DNA fragment which encodes a VP30 Ebola protein, said DNA fragment comprising the sequence specified in any of SEQ ID NO:4 and SEQ ID NO:7, or a polynucleotide fragment comprising at least 15 nucleotides.
 5. A DNA fragment which encodes a VP35 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO:5, or a polynucleotide fragment comprising at least 15 nucleotides.
 6. A DNA fragment which encodes a VP40 Ebola protein, said DNA fragment comprising the sequence specified in SEQ ID NO:6, or a polynucleotide fragment comprising at least 15 nucleotides.
 7. A DNA fragment which encodes a GP Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO:17 or a conservative substitution thereof.
 8. A DNA fragment which encodes a NP Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO:18 or a conservative substitution thereof.
 9. A DNA fragment which encodes a VP24 Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO:19 or a conservative substitution thereof.
 10. A DNA fragment which encodes a VP30 Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in any of SEQ ID NO:20 and SEQ ID NO:23 or a conservative substitution thereof.
 11. A DNA fragment which encodes a VP35 Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO:21 or a conservative substitution thereof.
 12. A DNA fragment which encodes a VP40 Ebola protein said DNA fragment comprising a DNA sequence encoding at least 5 amino acids specified in SEQ ID NO:22 or a conservative substitution thereof.
 13. A recombinant DNA construct comprising: (i) a vector, and (ii) at least one of the Ebola virus DNA fragments chosen from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6 and 7 or a fragment thereof comprising at least 15 nucleotides.
 14. A recombinant DNA construct comprising: (i) a vector, and (ii) at least one of the Ebola virus DNA fragments chosen from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24 and 25 or a conservative substitution thereof.
 15. The recombinant DNA construct of claim 13 wherein said DNA fragment induces a cytotoxic T lymphocyte, response or antibody response.
 16. The recombinant DNA construct of claim 14 wherein said DNA fragment induces a cytotoxic T lymphocyte response or antibody response.
 17. A recombinant DNA construct according to claim 13 wherein said vector is an expression vector.
 18. A recombinant DNA construct according to claim 13 wherein said vector is a prokaryotic vector.
 19. A recombinant DNA construct according to claim 13 wherein said vector is a eukaryotic vector.
 20. A recombinant DNA construct according to claim 14 wherein said vector is an expression vector.
 21. A recombinant DNA construct according to claim 14 wherein said vector is a prokaryotic vector.
 22. A recombinant DNA construct according to claim 14 wherein said vector is a eukaryotic vector.
 23. The recombinant DNA construct of claim 17 wherein said vector is a VEE virus replicon vector.
 24. The recombinant DNA construct of claim 20 wherein said vector is a VEE virus replicon vector.
 25. The recombinant DNA construct according to claim 23 wherein said Ebola virus DNA fragments are from Ebola Zaire
 1976. 26. The recombinant DNA construct according to claim 25 wherein said construct is VRepEboVP24.
 27. The recombinant DNA construct according to claim 25 wherein said construct is VRepEboVP30.
 28. The recombinant DNA construct according to claim 25 wherein said construct is VRepEboVP35.
 29. The recombinant DNA construct according to claim 25 wherein said construct is VRepEboVP40.
 30. The recombinant DNA construct according to claim 25 wherein said construct is for VRepEboNP.
 31. The recombinant DNA construct according to claim 25 wherein said construct is for VRepEboGP.
 32. The recombinant DNA construct according to claim 25 wherein said construct is for VRepEboVP30(#2).
 33. Self replicating RNA produced from a construct chosen from the group consisting of EboVP24ReP, EboVP30ReP, EboVP35ReP, EboVP40ReP, EboVPNPReP, EboVPGPReP, and EboVP30ReP(#2).
 34. Infectious alphavirus particles produced from packaging the self replicating RNA of claim
 33. 35. A pharmaceutical composition comprising infectious alphavirus particles according to claim 34 in an effective immunogenic amount in a pharmaceutically acceptable carrier and/or adjuvant.
 36. A host cell transformed with a recombinant DNA construct-according to claim
 13. 37. A host cell transformed with a recombinant DNA construct according to claim
 14. 38. A host cell according to claim 36 wherein said host cell is prokaryotic.
 39. A host cell according to claim 36 wherein said host cell is eukaryotic.
 40. A host cell according to claim 37 wherein said host cell is prokaryotic.
 41. A host cell according to claim 37 wherein said host cell is eukaryotic.
 42. A method for producing Ebola virus proteins comprising culturing the cells according to claim 36 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.
 43. A method for producing Ebola virus proteins comprising culturing the cells according to claim 37 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.
 44. A method for producing Ebola virus proteins comprising culturing the cells according to claim 38 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.
 45. A method for producing Ebola virus proteins comprising culturing the cells according to claim 39 under conditions such that said DNA fragment is expressed and said Ebola protein is produced.
 46. An isolated and purified Ebola GP protein specified in SEQ ID NO:17 and conservative substitutions thereof, or an immunologically identifiable portion thereof.
 47. An isolated and purified Ebola NP protein specified in SEQ ID NO:18 and conservative substitutions thereof or an immunologically identifiable portion thereof.
 48. An isolated and purified Ebola VP24 protein specified in SEQ ID NO:19 and conservative substitutions thereof or an immunologically identifiable portion thereof.
 49. An isolated and purified Ebola VP30 protein specified in any of SEQ ID NO:20 and SEQ ID NO:23 and conservative substitutions thereof or an immunologically identifiable portion thereof.
 50. An isolated and purified Ebola VP35 protein specified in SEQ ID NO:21 and conservative substitutions thereof or an immunologically identifiable portion thereof.
 51. An isolated and purified Ebola VP40 protein specified in SEQ ID NO:22 and conservative substitutions thereof or an immunologically identifiable portion thereof.
 52. An antibody to a peptide encoded by the sequence specified in SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, and
 25. 53. A method for detecting Ebola virus infection comprising contacting a sample from a subject suspected of having Ebola virus infection with a antibody according to claim 52 and detecting the presence or absence by detecting the presence or absence of a complex formed between the Ebola protein and antibodies specific therefor.
 54. A method for detecting the presence or absence of Ebola virus GP RNA in a sample using the polymerase chain reaction using primers for Ebola GP nucleic acid sequence specified in SEQ ID NO:1 for GP.
 55. An Ebola infection diagnostic kit comprising at least 12 consecutive nucleotides of SEQ ID NO:1 specific for the amplification of DNA or RNA of Ebola virus in a sample using the polymerase chain reaction and ancillary reagents suitable for use in such a reaction for detecting the presence or absence of Ebola virus DNA or RNA in a sample.
 56. A vaccine for Ebola comprising alphavirus particles of claim
 34. 57. A method for the diagnosis of Ebola virus infection comprising the steps of: (i) contacting a sample from an individual suspected of having Ebola virus infection with an antibody to Ebola proteins according to claim 52; and (ii) detecting the presence or absence of Ebola virus infection by detecting the presence or absence of a complex formed between Ebola proteins and antibodies specific therefor.
 58. A pharmaceutical composition comprising the self replicating RNA of claim 33 in an effective immunogenic amount in a pharmaceutically acceptable carrier and/or adjuvant.
 59. A pharmaceutical composition comprising one or more recombinant DNA constructs chosen from the group consisting of VRepEboVP24, VRepEboVP30, VRepEboVP35, VRepEboVP40, VRepEboNP, VRepEboGP, and VRepEboVP30(#2), in a pharmaceutically acceptable amount, in a pharmaceutically accpetable carrier and/or adjuvant.
 60. A pharmaceutical composition comprising comprising a peptide encoded by any of SEQ ID NO:24 and SEQ ID NO:25, in a pharmaceutically acceptable amount, in a pharmaceutically acceptable carrier and/or adjuvant. 